ANTI-Ly6E ANTIBODIES AND IMMUNOCONJUGATES AND METHODS OF USE

ABSTRACT

The invention provides anti-Ly6E antibodies, immunoconjugates and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/119,835, which is a national stage entry of International ApplicationNo. PCT/US2013/041848, filed 20 May 2013, which claims the benefit ofU.S. Provisional Application No. 61/649,775 filed on 21 May 2012, eachof which is fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to anti-Ly6E antibodies andimmunoconjugates and methods of using the same.

BACKGROUND

Lymphocyte antigen 6 complex, locus E (Ly6E), also known as retinoicacid induced gene E (RIG-E) and stem cell antigen 2 (SCA-2). It is a GPIlinked, 131 amino acid length, ˜8.4 kDa protein of unknown function withno known binding partners. It was initially identified as a transcriptexpressed in immature thymocyte, thymic medullary epithelial cells inmice. RIG-E, a human homolog of the murine Ly-6 family, is induced byretinoic acid during the differentiation of acute promyelocytic leukemiacell. Mao M., Yu M., Tong J.-H., Ye J., Zhu J., Huang Q.-H., Fu G., YuL., Zhao S.-Y., Waxman S., Lanotte M., Wang Z.-Y., Tan J.-Z., ChanS.-J., Chen Z. Proc. Natl. Acad. Sci. U.S.A. 93:5910-5914 (1996).

There is a need in the art for agents that target Ly6E for the diagnosisand treatment of Ly6E-associated conditions, such as cancer. Theinvention fulfills that need and provides other benefits.

SUMMARY OF THE INVENTION

The invention provides anti-Ly6E antibodies, immunoconjugates andmethods of using the same.

In some embodiments, an isolated antibody that binds to Ly6E isprovided. In other embodiments the antibody that binds to Ly6E binds toan epitope within amino acids 21-131 of SEQ ID NO:1. In anotherembodiment, such an antibody binds Ly6E with an affinity of ≦4 nM asmeasured by scatchard analysis. In another embodiment, such an antibodybinds Ly6E with an affinity of ≦7 nM as measured by surface plasmonresonance (SPR). In yet another embodiment, such an antibody is used asa medicament.

In one embodiment, an antibody that binds to an epitope within aminoacids 21-131 of SEQ ID NO:1 binds with an affinity of ≦4 nM as measuredby scatchard analysis, or binds Ly6E with an affinity of ≦7 nM asmeasured by SPR is a monoclonal antibody. In another embodiment, such anantibody is a human, humanized, or chimeric antibody.

In one embodiment, an antibody that binds to an epitope within aminoacids 21-131 of SEQ ID NO:1 binds with an affinity of ≦4 nM as measuredby scatchard analysis, and is internalized in a Ly6E-expressing cellupon binding to said epitope within amino acids 21-131 of SEQ ID NO: 1.In another embodiment, an antibody that binds to an epitope within aminoacids 21-131 of SEQ ID NO:1 binds Ly6E with an affinity of ≦7 nM asmeasured by SPR and is internalized in a Ly6E-expressing cell uponbinding to said epitope within amino acids 21-131 of SEQ ID NO:1.

In one embodiment, an antibody that binds to an epitope within aminoacids 21-131 of SEQ ID NO:1 binds with an affinity of ≦4 nM as measuredby scatchard analysis or binds Ly6E with an affinity of ≦7 nM asmeasured by SPR, wherein the antibody comprises (a) HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:12, (b) HVR-L3 comprising the aminoacid sequence of SEQ ID NO:9, and (c) HVR-H2 comprising the amino acidsequence of SEQ ID NO:11. In another embodiment, such an antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:10,(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:11, and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:12. In yetanother embodiment, the antibody described above further comprises (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:7; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:8; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:9. In yet anotherembodiment, such an antibody comprises (a) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO:8; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:9.

In one embodiment, for an antibody comprising (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:10, (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:11, and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:12 and (d) HVR-H1 comprising the amino acidsequence of SEQ ID NO:10, (e) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:11, and (f) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:12, it further comprises a light chain variable domainframework FR2 sequence of SEQ ID NO:20 or light chain variable domainframework FR3 of SEQ ID NO:21 or heavy chain variable domain frameworkFR1 or SEQ ID NO:23, or heavy chain variable domain framework FR2 of SEQID NO:24.

In one embodiment, an antibody that binds to an epitope within aminoacids 21-131 of SEQ ID NO:1 binds with an affinity of ≦4 nM as measuredby scatchard analysis or binds Ly6E with an affinity of ≦7 nM asmeasured by SPR, and comprises (a) a VH sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:5; (b) a VLsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:3; or (c) a VH sequence as in (a) and a VLsequence as in (b). In another embodiment, such an antibody comprises aVH sequence of SEQ ID NO:5. In yet another embodiment, such an antibodyfurther comprises a VL sequence of SEQ ID NO:3.

In one embodiment, an antibody that binds to an epitope within aminoacids 21-131 of SEQ ID NO:1 binds with an affinity of ≦4 nM as measuredby scatchard analysis, comprises a VH sequence of SEQ ID NO:5 and a VLsequence of SEQ ID NO:3. In another embodiment, an antibody that bindsto an epitope within amino acids 21-131 of SEQ ID NO:1 binds with anaffinity of ≦7 nM as measured by SPR, comprises a VH sequence of SEQ IDNO:5 and a VL sequence of SEQ ID NO:3.

In one embodiment, an antibody that binds to an epitope within aminoacids 21-131 of SEQ ID NO:1 binds with an affinity of ≦4 nM as measuredby scatchard analysis, which is an IgG1, IgG2a or IgG2b. In oneembodiment, an antibody that binds to an epitope within amino acids21-131 of SEQ ID NO:1 binds with an affinity of ≦7 nM as measured bySPR, which is an IgG1, IgG2a or IgG2b.

In one embodiment, isolated nucleic acids encoding an antibody asdescribed herein is provided. In another embodiment, a host cellcomprising such an isolated nucleic acid is also provided. In yetanother embodiment, a method of producing an antibody comprisingculturing the host cell comprising an isolated nucleic acid as describedherein so that the antibody is produced is also provided.

In one embodiment, an immunoconjugate comprising an antibody asdescribed herein and a cytotoxic agent is provided. In anotherembodiment, such an immunoconjugate has the formula Ab-(L-D)p, wherein:(a) Ab is an antibody as described herein; (b) L is a linker; (c) D is adrug selected from a maytansinoid, an auristatin, a calicheamicin, apyrrolobenzodiazepine, and a nemorubicin derivative; and (d) p rangesfrom 1-8. In some embodiments, such an immunoconjugate has D as anauristatin. In other embodiments, such an immunoconjugate has D havingformula D_(E):

wherein R² and R⁶ are each methyl, R³ and R⁴ are each isopropyl, R⁵ isH, R⁷ is sec-butyl, each R⁸ is independently selected from CH₃, O—CH₃,OH, and H; R⁹ is H; and R¹⁸ is —C(R⁸)₂—C(R⁸)₂-aryl.

In other embodiments, an immunoconjugate described herein, has as itsdrug, MMAE having the structure:

In one embodiment, the immunoconjugate described herein, has D as apyrrolobenzodiazepine of

Formula A:

wherein the dotted lines indicate the optional presence of a double bondbetween C1 and C2 or C2 and C3; R² is independently selected from H, OH,═O, ═CH₂, CN, R, OR, ═CH—R^(D), ═C(R^(D))₂, O—SO₂—R, CO₂R and COR, andoptionally further selected from halo or dihalo, wherein R^(D) isindependently selected from R, CO₂R, COR, CHO, CO₂H, and halo; R⁶ and R⁹are independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo; R⁷ is independently selected from H, R, OH, OR, SH,SR, NH₂, NHR, NRR′, NO₂, Me₃Sn and halo; Q is independently selectedfrom O, S and NH; R¹¹ is either H, or R or, where Q is O, SO₃M, where Mis a metal cation; R and R′ are each independently selected fromoptionally substituted C₁₋₈ alkyl, C₃₋₈ heterocyclyl and C₅₋₂₀ arylgroups, and optionally in relation to the group NRR′, R and R′ togetherwith the nitrogen atom to which they are attached form an optionallysubstituted 4-, 5-, 6- or 7-membered heterocyclic ring; R¹², R¹⁶, R¹⁹and R¹⁷ are as defined for R², R⁶, R⁹ and R⁷ respectively; R″ is a C₃₋₁₂alkylene group, which chain may be interrupted by one or moreheteroatoms and/or aromatic rings that are optionally substituted; and Xand X′ are independently selected from O, S and N(H). In someembodiments, the immunoconjugate as described herein, D has thestructure:

wherein n is 0 or 1.

In one embodiment, the immunoconjugate as described herein, D is anemorubicin derivative. In another embodiment, D has a structureselected from:

In one embodiment, an immunoconjugate as described herein comprises alinker that is cleavable by a protease. In other embodiments, the linkercomprises a val-cit dipeptide or a Phe-Lys dipeptide. In yet anotherembodiment, an immunoconjugate comprises a linker that is acid-labile.In other embodiments, the linker comprises hydrazone.

In one embodiment, an immunoconjugate has a formula selected from:

wherein S is a sulfur atom;

In some embodiments, p ranges from 2-5.

In one embodiment, an immunoconjugate of the invention as describedherein comprises an antibody comprising (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO:10, (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO:11, (c) HVR-H3 comprising the amino acid sequenceof SEQ ID NO:12, (d) HVR-L1 comprising the amino acid sequence of SEQ IDNO:7; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:9. In anotherembodiment, an immunoconjugate of the invention as described hereincomprises a VH sequence of SEQ ID NO:5 and a VL sequence of SEQ ID NO:3.

In one embodiment, a pharmaceutical formulation comprising theimmunoconjugate of the invention as described herein and apharmaceutically acceptable carrier is provided. In some embodiments, apharmaceutical formulation further comprises an additional therapeuticagent. In some embodiments, the additional therapeutic agent is achemotherapeutic agent, such as, for example, a platinum complex.

In one embodiment, methods of treating an individual having aLy6E-positive cancer are provided. In some embodiments, such methodscomprise administering a pharmaceutical formulation comprising animmunoconjugate of the invention as described herein comprising anantibody that binds Ly6E, e.g., as described herein. In otherembodiments, the Ly6E-positive cancer is selected from breast cancer,pancreatic cancer, colon cancer, colorectal cancer, melanoma, ovariancancer, non-small cell lung cancer, or gastric cancer. In someembodiments, a method of the invention comprises administering anadditional therapeutic agent to the individual. In some embodiments, theadditional therapeutic agent is a chemotherapeutic agent, such as, forexample, a platinum complex.

In some embodiments, methods of inhibiting proliferation of aLy6E-positive cell are provided. In one embodiment, such methodscomprise exposing a Ly6E-positive cell to an immunoconjugate of theinvention as described herein comprising an antibody that binds Ly6Eunder conditions permissive for binding of the immunoconjugate to Ly6Eon the surface of the cell. In some embodiments, an antibody that bindsLy6E is an antibody as described herein. In some embodiments,proliferation of the Ly6E-positive cell is thereby inhibited. In someembodiments, the cell is a breast cancer cell, pancreatic cancer cell,colon cancer cell, colorectal cancer cell, melanoma cell, ovarian cancercell, non-small cell lung cancer cell, or gastric cancer cell.

In one embodiment, an antibody that binds Ly6E as described herein isconjugated to a label. In some embodiments, such a label is a positronemitter. In some embodiments, the positron emitter is ⁸⁹Zr.

In some embodiments, a method of detecting human Ly6E in a biologicalsample is provided. In some embodiments, such a method comprisescontacting the biological sample with an anti-Ly6E antibody underconditions permissive for binding of the anti-Ly6E antibody to anaturally occurring human Ly6E, and detecting whether a complex isformed between the anti-Ly6E antibody and a naturally occurring humanLy6E in the biological sample. In some embodiments, an anti-Ly6Eantibody is an antibody described herein. In some embodiments, thebiological sample is a breast cancer sample, pancreatic cancer sample,colon cancer sample, colorectal cancer sample, melanoma sample, ovariancancer sample, non-small cell lung cancer sample, or gastric cancersample.

In one embodiment, a method for detecting a Ly6E-positive cancer isprovided. In such embodiments, a method comprises (i) administering alabeled anti-Ly6E antibody to a subject having or suspected of having aLy6E-positive cancer, and (ii) detecting the labeled anti-Ly6E antibodyin the subject, wherein detection of the labeled anti-Ly6E antibodyindicates a Ly6E-positive cancer in the subject. In some embodiments, ananti-Ly6E antibody is an antibody described herein. In otherembodiments, the Ly6E-positive cancer is selected from breast cancer,pancreatic cancer, colon cancer, colorectal cancer, melanoma, ovariancancer, non-small cell lung cancer, or gastric cancer. In one embodimentof the method described herein, an antibody that binds Ly6E as describedherein is conjugated to a label. In some embodiments, such a label is apositron emitter. In some embodiments, the positron emitter is ⁸⁹Zr.

In some embodiments, an isolated antibody that binds to Ly6E is providedfor use as a medicament. In other embodiments the antibody that binds toLy6E binds to an epitope within amino acids 21-131 of SEQ ID NO:1. Inanother embodiment, such an antibody binds Ly6E with an affinity of ≦4nM as measured by scatchard analysis. In another embodiment, such anantibody binds Ly6E with an affinity of ≦7 nM as measured by SPR. In yetanother embodiment, such an antibody is used for treating aLy6E-positive cancer. In some embodiments, such an antibody is used forinhibiting the proliferation of a Ly6E-positive cancer cell. In someembodiments, the Ly6E-positive cancer cell is a breast cancer cell,pancreatic cancer cell, colon cancer cell, colorectal cancer cell,melanoma cell, ovarian cancer cell, non-small cell lung cancer cell, orgastric cancer cell. In some embodiments, use of such an antibody asdescribed herein is used in the manufacture of a medicament. In anotherembodiment, such use is for a medicament for the treatment of aLy6E-positive cancer. In yet another embodiment, such use is forinhibiting the proliferation of a Ly6E-positive cancer cell. In someembodiments, the Ly6E-positive cancer cell is a breast cancer cell,pancreatic cancer cell, colon cancer cell, colorectal cancer cell,melanoma cell, ovarian cancer cell, non-small cell lung cancer cell, orgastric cancer cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a sequence alignment comparing the sequences of Ly6Eorthologs from human (SEQ ID NO: 1), Cynomolgus monkey (SEQ ID NO: 2),Rhesus (SEQ ID NO: 35), mouse (SEQ ID NO: 36), and rat species (SEQ IDNO: 37). The percent identity at the amino acid level between thesesequences in the extracellular domain (ECD) is shown to be ˜96% betweenhuman and cynomolgus monkey Ly6E and ˜52% between human and rat Ly6E.

FIG. 2 shows a Genelogic profile of Ly6E mRNA expression as furtherdescribed in Example 1. Measurements were carried out on the AffymetrixU133P chip and are expressed as scaled average differences in Ly6Eexpression in human tissues. Each dot represents a normal (green), tumor(red), or diseased non-tumor (blue) human tissue specimen. Rectanglesencompass the 25 to 75 percentile range for each distribution. WBC=whiteblood cells. Over-expression of Ly6E is seen in breast, pancreatic,colorectal, lung, melanoma and ovarian cancers, among others.

FIG. 3 a depicts QRT-PCR relative fold change in Ly6E transcriptexpression normalized to expression of RPL19 control gene in a panel ofnormal human tissues and select cancer cell lines and tissues asdescribed in Example 2. The results indicate that the Ly6E transcriptexpression in normal tissues is low compared to expression of Ly6E inbreast and pancreatic cancers.

FIG. 4 shows the light chain variable domain sequence alignment of ahumanized anti-Ly6E antibody (hu9B12.v12) as compared to a chimericanti-Ly6E antibody (xLy6E mu9B12) and a human kappa I consensus sequence(Kappa I consensus) Amino acid positions that differ from the humanconsensus frameworks are shaded in grey. Regions that were transferredto generate the CDR graft are boxed. Positions are numbered according toKabat.

FIG. 5 shows the heavy chain variable domain sequence alignment of ahumanized anti-Ly6E antibody (hu9B12.v12) as compared to a chimericanti-Ly6E antibody (xLy6E mu9B12) and a human VH₂ consensus sequence(VH2 consensus) Amino acid positions that differ from the humanconsensus frameworks are shaded in grey. Regions that were transferredto generate the CDR graft are boxed. Positions are numbered according toKabat.

FIG. 6 shows the heavy chain variable domain sequence alignment of ahumanized anti-Ly6E antibody (hu9B12.v12) as compared to a chimericanti-Ly6E antibody (xLy6E mu9B12) and a human VH₃ consensus sequence(VH3 consensus) Amino acid positions that differ from the humanconsensus frameworks are shaded in grey. Regions that were transferredto generate the CDR graft are boxed. Positions are numbered according toKabat.

FIGS. 7A-7C show the in vivo efficacy of an anti-Ly6E ADC in a xenograftmouse model as described in Example 7. FIG. 7A shows subcutaneous tumorsestablished in immunodeficient mice inoculated with HCC1569 X2 breastcancer cells. When tumor volumes reached approximately 100-250 mm³ (day0), animals were given a single IV injection of either control ADC(Control-vc-MMAE) or a humanized anti-Ly6E (hu9B12 v12) ADC(MC-vc-PAB-MMAE) at the indicated doses. Average tumor volumes withstandard deviations were determined from 9 animals per groups (indicatedon graph). FIG. 7B shows surface Ly6E protein expression in live HCC1569X2 cells as seen by flow cytometry, where the gray peak indicates cellstreated to secondary detection reagent alone and the black peakindicates cells treated with 3 μg/mL Ly6E antibody (hu9B12 v12) ADCfollowed by treatment with Alexafluor 488 conjugated to Human IgG as asecondary detection reagent. Expression of Ly6E as a GeoMean value isshown to the right of the histogram. FIG. 7C shows cell killing byhu9B12 v12 ADC titration for the breast cancer cell line HCC1569 X2. Theindicated concentrations of hu9B12 v12 ADC, control IgG-vc-MMAE, orequivalent amount of PBS vehicle control were incubated with cells for 5days and relative cell viability (y-axis) assessed using CellTiter-Glo.

FIGS. 8A-8D show the in vivo efficacy of an anti-Ly6E ADC in a xenograftmouse model. FIG. 8A shows subcutaneous tumors established inimmunodeficient mice inoculated with SU.86.86 pancreatic cancer cells.When tumor volumes reached approximately 100-250 mm³ (day 0), animalswere given a single IV injection of either control ADC (Control-vc-MMAE)or anti-Ly6E ADC at the indicated doses. Average tumor volumes withstandard deviations were determined from 9 animals per groups (indicatedon graph). FIG. 8B compares total Ly6E protein expression in HCC1569 X1and SU.86.86 cell lysates by immunoblotting. Total β-Actin proteinlevels were measured in parallel to serve as loading controls. FIG. 8Cshows cell killing by anti-Ly6E ADC titration for the pancreatic cancercell line SU.86.86. The indicated concentrations of anti-Ly6E ADC,control IgG-vc-MMAE, or equivalent amount of PBS vehicle control wereincubated with cells for 5 days and relative cell viability (y-axis)assessed using CellTiter-Glo. FIG. 8D shows 1+ Ly6E staining on SU.86.86cell pellet by immunohistochemistry.

FIGS. 9A-9C show the in vivo efficacy of anti-Ly6E ADC in primary breastcancer tumor xenograft model HBCx-9 established at XenTech (Evry,France). FIG. 9A shows subcutaneous tumors established inimmunodeficient mice implanted with patient derived breast cancer tumormaterial. When tumor volumes reached approximately 100-250 mm³ (day 0),animals were given a single IV injection of either control ADC(Control-vc-MMAE) or anti-Ly6E ADC at the indicated doses. Average tumorvolumes with standard deviations were determined from 9 animals pergroups (indicated on graph). FIG. 9B compares total Ly6E proteinexpression in various XenTech primary tumor models and in HCC1569 X1 andSU.86.86 cell lysates by immunoblotting. Total β-Actin protein levelswere measured in parallel to serve as loading controls. FIG. 9C showsLy6E staining on HBCx-9 tumors by immunohistochemistry. Independentstaining of multiple tumor samples showed heterogeneous stainingpatterns. The percent of tumor cells staining at a 1+ level for Ly6E isindicated.

FIGS. 10A-10C show the in vivo efficacy of anti-Ly6E ADC in primarybreast cancer tumor xenograft model HBCx-8 established at XenTech (Evry,France). FIG. 10A shows subcutaneous tumors established inimmunodeficient mice implanted with patient derived breast cancer tumormaterial. When tumor volumes reached approximately 100-250 mm³ (day 0),animals were given a single IV injection of either control ADC(Control-vc-MMAE) or anti-Ly6E ADC at the indicated doses. Average tumorvolumes with standard deviations were determined from 10 animals pergroups (indicated on graph). FIG. 10B compares total Ly6E proteinexpression in various XenTech primary tumor models and in HCC1569 X1 andSU.86.86 cell lysates by immunoblotting. Total β-Actin protein levelswere measured in parallel to serve as loading controls. FIG. 10C showsLy6E staining on HBCx-8 tumors by immunohistochemistry. Independentstaining of multiple tumor samples showed heterogeneous stainingpatterns at a 1+ or 2+ level.

FIGS. 11A-11C show the in vivo efficacy of anti-Ly6E ADC in primarybreast cancer tumor xenograft model MAXF-1162 established at OncotestGmbH (Freiburg, Germany). FIG. 11A shows subcutaneous tumors establishedin immunodeficient mice implanted with patient derived breast cancertumor material. When tumor volumes reached approximately 100-250 mm³(day 0), animals were given a single IV injection of either control ADC(Control-vc-MMAE) or anti-Ly6E ADC at the indicated doses. Average tumorvolumes with standard deviations were determined from 10 animals pergroups (indicated on graph). FIG. 11B compares total Ly6E proteinexpression in various Oncotest primary tumor models and cell lysates byimmunoblotting. Total GAPDH protein levels were measured in parallel toserve as loading controls. FIG. 11C shows Ly6E staining on MAXF-1162tumors by immunohistochemistry. Independent staining of multiple tumorsamples showed heterogeneous staining patterns. The percent of tumorcells staining at a 1+ or 2+ level for Ly6E is indicated.

FIGS. 12A-12C show the in vivo efficacy of anti-Ly6E ADC in primarypancreatic cancer tumor xenograft model PAXF-1657 established atOncotest GmbH (Freiburg, Germany). FIG. 12A shows subcutaneous tumorsestablished in immunodeficient mice with patient derived pancreaticcancer tumor explants. When tumor volumes reached approximately 100-250mm³ (day 0), animals were given a single IV injection of either controlADC (Control-vc-MMAE) or anti-Ly6E ADC at the indicated doses. Averagetumor volumes with standard deviations were determined from 10 animalsper groups (indicated on graph). FIG. 12B compares total Ly6E proteinexpression in various Oncotest primary tumor models and cell lysates byimmunoblotting. Total GAPDH protein levels were measured in parallel toserve as loading controls. FIG. 12C shows Ly6E staining on PAXF-1657tumors by immunohistochemistry. Independent staining of multiple tumorsamples showed heterogeneous staining patterns. The percent of tumorcells staining at a very weak (+/−) level for Ly6E is indicated.

FIGS. 13A-D compare the in vivo efficacy of various anti-Ly6E ADCconjugates in mouse xenograft model. FIGS. 13A, 13B and 13C all showsubcutaneous tumors established in immunodeficient mice with pancreaticcancer cell line SU.86.86. When tumor volumes reached approximately100-250 mm³ (day 0), animals were given a single IV injection of 1 mpkof either control ADC or anti-Ly6E ADC as indicated on the graphs.Average tumor volumes with standard deviations were determined from 10animals per groups (indicated on graphs). FIG. 13D shows 1+ Ly6Estaining on SU.86.86 cell pellet by immunohistochemistry.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. DEFINITIONS

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The terms “anti-Ly6E antibody” and “an antibody that binds to Ly6E”refer to an antibody that is capable of binding Ly6E with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting Ly6E. In one embodiment, the extent ofbinding of an anti-Ly6E antibody to an unrelated, non-Ly6E protein isless than about 10% of the binding of the antibody to Ly6E as measured,e.g., by a radioimmunoassay (RIA) or by scatchard analysis or by surfaceplasmon resonance, such as, for example, Biacore. In certainembodiments, an antibody that binds to Ly6E has a dissociation constant(Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM(e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to10⁻¹³ M). In certain embodiments, an anti-Ly6E antibody binds to anepitope of Ly6E that is conserved among Ly6E from different species.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

The term “antibody drug conjugate” (ADC) as used herein is equivalent tothe term “immunoconjugate”.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include a cancer that over-expressesLy6E, which may include, for example, breast cancer and/or metastaticbreast cancer, including Her2 negative breast cancers and/or triplenegative breast cancers, pancreatic cancer, colon cancer, colorectalcancer, melanoma, ovarian cancer, non-small cell lung cancer (eithersquamous and/or non-squamous), gastric cancer, squamous cell cancer,small-cell lung cancer, adenocarcinoma of the lung, squamous carcinomaof the lung, cancer of the peritoneum, hepatocellular cancer,gastrointestinal cancer, glioma, cervical cancer, liver cancer, bladdercancer, hepatoma, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, leukemia and otherlymphoproliferative disorders, and various types of head and neckcancer.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “epitope” refers to the particular site on an antigen moleculeto which an antibody binds.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain However, the C-terminal lysine (Lys447) of the Fc region mayor may not be present. Unless otherwise specified herein, numbering ofamino acid residues in the Fc region or constant region is according tothe EU numbering system, also called the EU index, as described in Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent. An immunoconjugate is equivalent to the term “antibody drugconjugate” (ADC).

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-Ly6E antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “Ly6E,” as used herein, refers to any native, mature Ly6E whichresults from processing of a Ly6E precursor protein in a cell. The termincludes Ly6E from any vertebrate source, including mammals such asprimates (e.g. humans and cynomolgus or rhesus monkeys) and rodents(e.g., mice and rats), unless otherwise indicated. The term alsoincludes naturally occurring variants of Ly6E, e.g., splice variants orallelic variants. The amino acid sequence of an exemplary human Ly6Eprecursor protein, with signal sequence (amino acids 1-20=signalsequence) is shown in SEQ ID NO: 1. The amino acid sequence of anexemplary mature human Ly6E is shown in SEQ ID NO: 38. The sequence foramino acids 1-131 of an exemplary cynomolgous monkey Ly6E is shown inSEQ ID NO: 2. The amino acid sequence of an exemplary maturecynomologous Ly6E is shown in SEQ ID NO: 39. The amino acid sequence foran exemplary rat Ly6E precursor (with signal sequence, amino acids 1-26)and mature sequences are shown in SEQ ID NOs: 37 and 42, respectively.The amino acid sequences for exemplary mouse Ly6E precursor (with signalsequence, amino acids 1-26) and mature sequences are shown in SEQ IDNOs: 36 and 41, respectively.

The term “Ly6E-positive cancer” refers to a cancer comprising cells thatexpress Ly6E on their surface. For the purposes of determining whether acell expresses Ly6E on the surface, Ly6E mRNA expression is consideredto correlate to Ly6E expression on the cell surface. In someembodiments, expression of Ly6E mRNA is determined by a method selectedfrom in situ hybridization and RT-PCR (including quantitative RT-PCR).Alternatively, expression of Ly6E on the cell surface can be determined,for example, using antibodies to Ly6E in a method such asimmunohistochemistry, FACS, etc. In some embodiments, a Ly6E-positivecancer means a breast cancer, metastatic breast cancer, including Her2negative breast cancers and/or triple negative breast cancers,pancreatic cancer, colon cancer, colorectal cancer, melanoma, ovariancancer, non-small cell lung cancer (either squamous and/ornon-squamous), or gastric cancer, each of which that exhibits a highlevel of Ly6E expression.

The term “Ly6E-positive cell” refers to a cancer cell that expressesLy6E on its surface.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH_(1,) CH₂, andCH₃). Similarly, from N- to C-terminus, each light chain has a variableregion (VL), also called a variable light domain or a light chainvariable domain, followed by a constant light (CL) domain. The lightchain of an antibody may be assigned to one of two types, called kappa(κ) and lambda (λ), based on the amino acid sequence of its constantdomain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary. In situations where ALIGN-2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

A “platinum complex” as used herein refers to anti-cancer chemotherapydrugs such as, for example, but not limited to, cisplatin, oxaliplatin,carboplatin, iproplatin, satraplatin, CI-973, AZ0473, DWA2114R,nedaplatin, and sprioplatin, which exert efficacy against tumors basedon their ability to covalently bind to DNA.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W. H. Freeman and Co., page 91(2007)). A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

“Alkyl” is C₁-C₁₈ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Examples are methyl (Me, —CH₃), ethyl (Et,—CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃.

The term “C₁-C₈ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 8 carbonatoms. Representative “C₁-C₈ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl,-n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while branched C₁-C₈ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C₁-C₈ alkylsinclude, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl,-isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl,-2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl,-acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl,-2-pentynyl, -3-methyl-1 butynyl. A C₁-C₈ alkyl group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where eachR′ is independently selected from H, —C₁-C₈ alkyl and aryl.

The term “C₁-C₁₂ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 12carbon atoms. A C₁-C₁₂ alkyl group can be unsubstituted or substitutedwith one or more groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

The term “C₁-C₆ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 6 carbonatoms. Representative “C₁-C₆ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -and n-hexyl; whilebranched C₁-C₆ alkyls include, but are not limited to, -isopropyl,-sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl;unsaturated C₁-C₆ alkyls include, but are not limited to, -vinyl,-allyl, -1-butenyl, -2-butenyl, and -isobutylenyl, -1-pentenyl,-2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl,-2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, and 3-hexyl. A C₁-C₆ alkylgroup can be unsubstituted or substituted with one or more groups, asdescribed above for C₁-C₈ alkyl group.

The term “C₁-C₄ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 4 carbonatoms. Representative “C₁-C₄ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl; while branched C₁-C₄ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl; unsaturated C₁-C₄ alkyls include, but are not limited to,-vinyl, -allyl, -1-butenyl, -2-butenyl, and -isobutylenyl. A C₁-C₄ alkylgroup can be unsubstituted or substituted with one or more groups, asdescribed above for C₁-C₈ alkyl group.

“Alkoxy” is an alkyl group singly bonded to an oxygen. Exemplary alkoxygroups include, but are not limited to, methoxy (—OCH₃) and ethoxy(—OCH₂CH₃). A “C₁-C₅ alkoxy” is an alkoxy group with 1 to 5 carbonatoms. Alkoxy groups may can be unsubstituted or substituted with one ormore groups, as described above for alkyl groups.

“Alkenyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond. Examples include, but are not limitedto: ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl(—C₅H₇), and 5-hexenyl (—CH₂ CH₂CH₂CH₂CH═CH₂). A “C₂-C₈ alkenyl” is ahydrocarbon containing 2 to 8 normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond.

“Alkynyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond. Examples include, but are not limited to:acetylenic (—C≡CH) and propargyl (—CH₂C≡CH). A “C₂-C₈ alkynyl” is ahydrocarbon containing 2 to 8 normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond.

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—) 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

A “C₁-C₁₀ alkylene” is a straight chain, saturated hydrocarbon group ofthe formula —(CH₂)₁₋₁₀—. Examples of a C₁-C₁₀ alkylene includemethylene, ethylene, propylene, butylene, pentylene, hexylene,heptylene, ocytylene, nonylene and decalene.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to: 1,2-ethylene(—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to: acetylene (—C≡C—),propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡C—).

“Aryl” refers to a carbocyclic aromatic group. Examples of aryl groupsinclude, but are not limited to, phenyl, naphthyl and anthracenyl. Acarbocyclic aromatic group or a heterocyclic aromatic group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′,—OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₅-C₂₀ aryl” is an aryl group with 5 to 20 carbon atoms in thecarbocyclic aromatic rings. Examples of C₅-C₂₀ aryl groups include, butare not limited to, phenyl, naphthyl and anthracenyl. A C₅-C₂₀ arylgroup can be substituted or unsubstituted as described above for arylgroups. A “C₅-C₁₄ aryl” is an aryl group with 5 to 14 carbon atoms inthe carbocyclic aromatic rings. Examples of C₅-C₁₄ aryl groups include,but are not limited to, phenyl, naphthyl and anthracenyl. A C₅-C₁₄ arylgroup can be substituted or unsubstituted as described above for arylgroups.

An “arylene” is an aryl group which has two covalent bonds and can be inthe ortho, meta, or para configurations as shown in the followingstructures:

in which the phenyl group can be unsubstituted or substituted with up tofour groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂ —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1 and thelike. The arylalkyl group comprises 6 to 20 carbon atoms, e.g. the alkylmoiety, including alkanyl, alkenyl or alkynyl groups, of the arylalkylgroup is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbonatoms.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Typicalheteroarylalkyl groups include, but are not limited to,2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkylgroup comprises 6 to 20 carbon atoms, e.g. the alkyl moiety, includingalkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms and 1to 3 heteroatoms selected from N, O, P, and S. The heteroaryl moiety ofthe heteroarylalkyl group may be a monocycle having 3 to 7 ring members(2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), forexample a bicyclo [4,5], [5,5], [5,6], or [6,6] system.

“Substituted alkyl,” “substituted aryl,” and “substituted arylalkyl”mean alkyl, aryl, and arylalkyl respectively, in which one or morehydrogen atoms are each independently replaced with a substituent.Typical substituents include, but are not limited to, —X, —R, —O⁻, —OR,—SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO,—NO₂, ═N₂, —N₃, NC(═O)R, —C(═O)R, —C(═O)NR₂, —SO₃ ⁻, —SO₃H, —S(═O)₂R,—OS(═O)₂OR, —S(═O)₂NR, —S(═O)R, —OP(═O)(OR)₂, —P(═O)(OR)₂, —PO⁻ ₃,—PO₃H₂, —C(═O)R, —C(═O)X, —C(═S)R, —CO₂R, —C(═S)OR, —C(═O)SR, —C(═S)SR,—C(═O)NR₂, —C(═S)NR₂, —C(═NR)NR₂, where each X is independently ahalogen: F, Cl, Br, or I; and each R is independently —H, C₂-C₁₈ alkyl,C₆-C₂₀ aryl, C₃-C₁₄ heterocycle, protecting group or prodrug moiety.Alkylene, alkenylene, and alkynylene groups as described above may alsobe similarly substituted.

“Heteroaryl” and “heterocycle” refer to a ring system in which one ormore ring atoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 3 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example a bicyclo [4,5], [5,5], [5,6], or [6,6]system.

Exemplary heterocycles are described, e.g., in Paquette, Leo A.,“Principles of Modern Heterocyclic Chemistry” (W. A. Benjamin, New York,1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry ofHeterocyclic Compounds, A series of Monographs” (John Wiley & Sons, NewYork, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28;and J. Am. Chem. Soc. (1960) 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,13-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or I3-carboline. Still more typically, nitrogen bondedheterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl,1-pyrazolyl, and 1-piperidinyl.

A “C₃-C₈ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. Representative examples of a C₃-C₈ heterocycle include, but arenot limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl. A C₃-C₈ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₈ heterocyclo” refers to a C₃-C₈ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond. A C₃-C₈ heterocyclo can be unsubstituted or substituted with up tosix groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

A “C₃-C₂₀ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. A C₃-C₂₀ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₂₀ heterocyclo” refers to a C₃-C₂₀ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond.

“Carbocycle” means a saturated or unsaturated ring having 3 to 7 carbonatoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Monocycliccarbocycles have 3 to 6 ring atoms, still more typically 5 or 6 ringatoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g. arranged as abicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicyclo [5,6] or [6,6] system. Examples of monocycliccarbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl,and cyclooctyl.

A “C₃-C₈ carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated orunsaturated non-aromatic carbocyclic ring. Representative C₃-C₈carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl,-cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl,-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and-cyclooctadienyl. A C₃-C₈ carbocycle group can be unsubstituted orsubstituted with one or more groups including, but not limited to,-C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₃-C₈ carbocyclo” refers to a C₃-C₈ carbocycle group defined abovewherein one of the carbocycle groups' hydrogen atoms is replaced with abond.

“Linker” refers to a chemical moiety comprising a covalent bond or achain of atoms that covalently attaches an antibody to a drug moiety. Invarious embodiments, linkers include a divalent radical such as analkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as:—(CR₂)_(n)O(CR₂)_(n)—, repeating units of alkyloxy (e.g. polyethylenoxy,PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino,Jeffamine™); and diacid ester and amides including succinate,succinamide, diglycolate, malonate, and caproamide. In variousembodiments, linkers can comprise one or more amino acid residues, suchas valine, phenylalanine, lysine, and homolysine.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and 1 or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or 1 meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Certain leaving groups are well known in theart, and examples include, but are not limited to, a halide (e.g.,chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl(tosyl), trifluoromethylsulfonyl (triflate), andtrifluoromethylsulfonate.

The term “protecting group” refers to a substituent that is commonlyemployed to block or protect a particular functionality while reactingother functional groups on the compound. For example, an“amino-protecting group” is a substituent attached to an amino groupthat blocks or protects the amino functionality in the compound.Suitable amino-protecting groups include, but are not limited to,acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ)and 9-fluorenylmethylenoxycarbonyl (Fmoc). For a general description ofprotecting groups and their use, see T. W. Greene, Protective Groups inOrganic Synthesis, John Wiley & Sons, New York, 1991, or a lateredition.

II. COMPOSITIONS AND METHODS

In one aspect, the invention is based, in part, on antibodies that bindto LY6E and immunoconjugates comprising such antibodies. Antibodies andimmunoconjugates of the invention are useful, e.g., for the diagnosis ortreatment of LY6E-positive cancers.

A. Exemplary Anti-Ly6E Antibodies

In some embodiments, the invention provides isolated antibodies thatbind to LY6E. In certain embodiments, an anti-LY6E antibody has at leastone or more of the following characteristics, in any combination:

-   -   (a) binds to an epitope within amino acids 21-131 of SEQ ID NO:        1; and    -   (b) binds Ly6E with an affinity of ≦7 nM, or ≦6 nM, or ≦5 nM, or        ≦4 nM, or ≦3 nM, or ≦2 nM, or ≦1 nM, and optionally ≧0.0001 nM,        or ≧0.001 nM, or ≧0.01 nM as measured by either SPR or scatchard        analysis.

A nonlimiting exemplary antibody of the invention is the murine 9B12 asshown in FIGS. 4-6 and humanized variants thereof, such as, for example,hu9B12.v12, as shown in FIGS. 4-6. In some embodiments, Ly6E is humanLy6E. In some embodiments, Ly6E is selected from human, cynomolgusmonkey, rhesus monkey, mouse or rat Ly6E.

In some embodiments, an anti-Ly6E antibody binds to an epitope withinamino acids 21-131 of SEQ ID NO: 1. In some such embodiments, theanti-Ly6E antibody binds Ly6E with an affinity of ≦7 nM, or ≦6 nM, or ≦5nM, or ≦4 nM, or ≦3 nM, or ≦2 nM, or ≦1 nM, and optionally ≧0.0001 nM,or ≧0.001 nM, or ≧0.01 nM as measured by either SPR or scatchardanalysis. A nonlimiting exemplary antibody of the invention is themurine 9B12 as shown in FIGS. 4-6 and humanized variants thereof, suchas, for example, hu9B12.v12, as shown in FIGS. 4-6. In some embodiments,Ly6E is human Ly6E. In some embodiments, Ly6E is human Ly6E orcynomolgus monkey Ly6E.

In one aspect, the invention provides an anti-Ly6E antibody comprisingat least one, two, three, four, five, or six HVRs selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:10; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:11; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:12; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:7; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:8; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:9.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:10; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:11; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:12. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:12. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:12 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:9. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:12, HVR-L3 comprising the amino acid sequence of SEQ ID NO:9, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:11. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:10; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:11; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:12.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:7; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:8; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:9. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:7; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:8; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:9.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:10, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:11, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:12; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:7, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:8, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:9.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:10; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:11; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:12; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:7; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:8; and (f) HVR-L3 comprising anamino acid sequence selected from SEQ ID NO:9.

In certain embodiments, any one or more amino acids of an anti-Ly6Eantibody as provided above are substituted at the following HVRpositions:

In any of the above embodiments, an anti-Ly6E antibody is humanized Inone embodiment, an anti-Ly6E antibody comprises HVRs as in any of theabove embodiments, and further comprises an acceptor human framework,e.g. a human immunoglobulin framework or a human consensus framework. Inanother embodiment, an anti-Ly6E antibody comprises HVRs as in any ofthe above embodiments, and it further comprises a light chain variabledomain framework FR2 sequence of SEQ ID NO:20 or light chain variabledomain framework FR3 of SEQ ID NO:21 or heavy chain variable domainframework FR1 or SEQ ID NO:23, or heavy chain variable domain frameworkFR2 of SEQ ID NO:24.

In another aspect, an anti-Ly6E antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:5. In certain embodiments, a VH sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence, but an anti-Ly6Eantibody comprising that sequence retains the ability to bind to Ly6E.In certain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:5. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the HVRs (i.e., in the FRs). Optionally, the anti-Ly6E antibodycomprises the VH sequence in SEQ ID NO:5, including post-translationalmodifications of that sequence. In a particular embodiment, the VHcomprises one, two or three HVRs selected from: (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:10, (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:11, and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:12.

In another aspect, an anti-Ly6E antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:3. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-Ly6E antibody comprising that sequenceretains the ability to bind to Ly6E. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO:3. In certain embodiments, the substitutions, insertions, ordeletions occur in regions outside the HVRs (i.e., in the FRs).Optionally, the anti-Ly6E antibody comprises the VL sequence in SEQ IDNO:3, including post-translational modifications of that sequence. In aparticular embodiment, the VL comprises one, two or three HVRs selectedfrom (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:7; (b)HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:9.

In another aspect, an anti-Ly6E antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In one embodiment, theantibody comprises the VH and VL sequences in SEQ ID NO:5 and SEQ IDNO:3, respectively, including post-translational modifications of thosesequences.

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-Ly6E antibody provided herein. For example,in certain embodiments, an antibody is provided that binds to the sameepitope as an anti-Ly6E antibody comprising a VH sequence of SEQ ID NO:5and a VL sequence of SEQ ID NO:3. In certain embodiments, an antibody isprovided that binds to an epitope within a fragment of Ly6E consistingof amino acids 21-131 of SEQ ID NO:1.

In a further aspect of the invention, an anti-Ly6E antibody according toany of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In one embodiment, an anti-Ly6Eantibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody,or F(ab′)₂ fragment. In another embodiment, the antibody is a fulllength antibody, e.g., an intact IgG1 antibody or other antibody classor isotype as defined herein.

In a further aspect, an anti-Ly6E antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below.

Assays

To determine whether an anti-LY6E antibody “binds to an epitope withinamino acids 21-131 of SEQ ID NO: 1 Ly6E polypeptides with N- andC-terminal deletions are expressed in 293 cells and binding of theantibody to the truncated polypeptides is tested by FACS, wherein asubstantial reduction (≧70% reduction) or elimination of binding of theantibody to a truncated polypeptide relative to binding to full-lengthLy6E expressed in 293 cells indicates that the antibody does not bind tothat truncated polypeptide.

Whether an anti-Ly6E antibody “binds with an affinity of ≦6 nM, or ≦5nM, or ≦4 nM, or ≦3 nM, or ≦2 nM, or ≦1 nM,” is determined according toa scatchard analysis as described herein in Example 4. Alternatively, ananti-Ly6E antibody affinity can be determined according to, for example,a BIAcore assay. Specifically, Kd is measured using surface plasmonresonance assays using a BIACORE®-3000 (BIAcore, Inc., Piscataway,N.J.). BIAcore™ research grade CM5 chips are activated with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) andN-hydroxysuccinimide (NHS) reagents according to the supplier'sinstructions. Goat anti-human Fc IgGs are coupled to the chips toachieve approximately 10,000 response units (RU) in each flow cell.Unreacted coupling groups are blocked with 1M ethanolamine For kineticsmeasurements, anti-Ly6E antibodies are captured to achieve approximately300 RU. Two-fold serial dilutions of human Ly6E (for example, aminoacids 21-131 fused to His-Fc expressed in a baculovirus system, or aminoacids 21-131 fused to Fc expressed from CHO cells; 125 nM to 0.49 nM)are injected in HBS-P buffer (0.01M HEPES pH7.4, 0.15M NaCl, 0.005%surfactant P20) at 25° C. with a flow rate of 30 μl/min. Associationrates (k_(on)) and dissociation rates (k_(off)) are calculated using a1:1 Langmuir binding model (BIAcore™ Evaluation Software version 3.2).The equilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surfaceplasmon resonance assay above, then the on-rate can be determined byusing a fluorescent quenching technique that measures the increase ordecrease in fluorescence emission intensity (excitation=295 nm;emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophotometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

In any of the above embodiments, an anti-Ly6E antibody is humanized Inone embodiment, an anti-Ly6E antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaIV consensus (VL_(KIV)) framework and/or the VH framework VH₁.

In a further aspect of the invention, an anti-Ly6E antibody according toany of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In one embodiment, an anti-Ly6Eantibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody,or F(ab′)₂ fragment. In another embodiment, the antibody is asubstantially full length antibody, e.g., an IgG1 antibody or otherantibody class or isotype as defined herein.

In a further aspect, an anti-Ly6E antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below.

In a further aspect of the invention, an anti-Ly6E antibody according toany of the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-Ly6E antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG2a antibody or other antibody class or isotype asdefined herein.

In a further aspect, an anti-Ly6E antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below.

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≦104, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or≦0.001 nM, and optionally is ≧10⁻¹³ M. (e.g. 10⁻⁸M or less, e.g. from10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using scatchardanalysis, as described in Example 4. According to another embodiment, Kdis measured using surface plasmon resonance assays using a BIACORE®-2000or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CMS chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CMS, BIACORE, Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for LY6E and the other is for any other antigen. Incertain embodiments, one of the binding specificities is for LY6E andthe other is for CD3. See, e.g., U.S. Pat. No. 5,821,337. In certainembodiments, bispecific antibodies may bind to two different epitopes ofLy6E. Bispecific antibodies may also be used to localize cytotoxicagents to cells which express Ly6E. Bispecific antibodies can beprepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see,e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to Ly6E as well asanother, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex is usedto identify contact points between the antibody and antigen. Suchcontact residues and neighboring residues may be targeted or eliminatedas candidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH₂ domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example Asn297 refers to the asparagineresidue located at about position 297 in the Fc region (Eu numbering ofFc region residues); however, Asn297 may also be located about ±3 aminoacids upstream or downstream of position 297, i.e., between positions294 and 300, due to minor sequence variations in antibodies. Suchfucosylation variants may have improved ADCC function. See, e.g., USPatent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621(Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to“defucosylated” or “fucose-deficient” antibody variants include: US2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-LY6E antibody described herein isprovided. Such nucleic acid may encode an amino acid sequence comprisingthe VL and/or an amino acid sequence comprising the VH of the antibody(e.g., the light and/or heavy chains of the antibody). In a furtherembodiment, one or more vectors (e.g., expression vectors) comprisingsuch nucleic acid are provided. In a further embodiment, a host cellcomprising such nucleic acid is provided. In one such embodiment, a hostcell comprises (e.g., has been transformed with): (1) a vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and an amino acid sequence comprising the VH ofthe antibody, or (2) a first vector comprising a nucleic acid thatencodes an amino acid sequence comprising the VL of the antibody and asecond vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-LY6E antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-LY6E antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-LY6E antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, BIACore®, FACS,or Western blot.

In another aspect, competition assays may be used to identify anantibody that competes with any of the antibodies described herein forbinding to LY6E. In certain embodiments, such a competing antibody bindsto the same epitope (e.g., a linear or a conformational epitope) that isbound by an antibody described herein. Detailed exemplary methods formapping an epitope to which an antibody binds are provided in Morris(1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol.66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized LY6E is incubated in asolution comprising a first labeled antibody that binds to LY6E (e.g.,any of the antibodies described herein) and a second unlabeled antibodythat is being tested for its ability to compete with the first antibodyfor binding to LY6E. The second antibody may be present in a hybridomasupernatant. As a control, immobilized LY6E is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to LY6E, excess unbound antibody is removed, and theamount of label associated with immobilized LY6E is measured. If theamount of label associated with immobilized LY6E is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to LY6E. See Harlow and Lane (1988) Antibodies: A LaboratoryManual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-LY6Eantibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes(i.e., a radioconjugate).

Immunoconjugates allow for the targeted delivery of a drug moiety to atumor, and, in some embodiments intracellular accumulation therein,where systemic administration of unconjugated drugs may result inunacceptable levels of toxicity to normal cells (Polakis P. (2005)Current Opinion in Pharmacology 5:382-387).

Antibody-drug conjugates (ADC) are targeted chemotherapeutic moleculeswhich combine properties of both antibodies and cytotoxic drugs bytargeting potent cytotoxic drugs to antigen-expressing tumor cells(Teicher, B. A. (2009) Current Cancer Drug Targets 9:982-1004), therebyenhancing the therapeutic index by maximizing efficacy and minimizingoff-target toxicity (Carter, P. J. and Senter P. D. (2008) The CancerJour. 14(3):154-169; Chari, R. V. (2008) Acc. Chem. Res. 41:98-107.

The ADC compounds of the invention include those with anticanceractivity. In some embodiments, the ADC compounds include an antibodyconjugated, i.e. covalently attached, to the drug moiety. In someembodiments, the antibody is covalently attached to the drug moietythrough a linker. The antibody-drug conjugates (ADC) of the inventionselectively deliver an effective dose of a drug to tumor tissue wherebygreater selectivity, i.e. a lower efficacious dose, may be achievedwhile increasing the therapeutic index (“therapeutic window”).

The drug moiety (D) of the antibody-drug conjugates (ADC) may includeany compound, moiety or group that has a cytotoxic or cytostatic effect.Drug moieties may impart their cytotoxic and cytostatic effects bymechanisms including but not limited to tubulin binding, DNA binding orintercalation, and inhibition of RNA polymerase, protein synthesis,and/or topoisomerase. Exemplary drug moieties include, but are notlimited to, a maytansinoid, dolastatin, auristatin, calicheamicin,pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives,PNU-159682, anthracycline, duocarmycin, vinca alkaloid, taxane,trichothecene, CC1065, camptothecin, elinafide, and stereoisomers,isosteres, analogs, and derivatives thereof that have cytotoxicactivity. Nonlimiting examples of such immunoconjugates are discussed infurther detail below.

1. Exemplary Antibody-Drug Conjugates

An exemplary embodiment of an antibody-drug conjugate (ADC) compoundcomprises an antibody (Ab) which targets a tumor cell, a drug moiety(D), and a linker moiety (L) that attaches Ab to D. In some embodiments,the antibody is attached to the linker moiety (L) through one or moreamino acid residues, such as lysine and/or cysteine.

An exemplary ADC has Formula I:

Ab-(L-D)_(p)   Formula I

where p is 1 to about 20. In some embodiments, the number of drugmoieties that can be conjugated to an antibody is limited by the numberof free cysteine residues. In some embodiments, free cysteine residuesare introduced into the antibody amino acid sequence by the methodsdescribed herein. Exemplary ADC of Formula I include, but are notlimited to, antibodies that have 1, 2, 3, or 4 engineered cysteine aminoacids (Lyon, R. et al (2012) Methods in Enzym. 502:123-138). In someembodiments, one or more free cysteine residues are already present inan antibody, without the use of engineering, in which case the existingfree cysteine residues may be used to conjugate the antibody to a drug.In some embodiments, an antibody is exposed to reducing conditions priorto conjugation of the antibody in order to generate one or more freecysteine residues.

a) Exemplary Linkers

A “Linker” (L) is a bifunctional or multifunctional moiety that can beused to link one or more drug moieties (D) to an antibody (Ab) to forman antibody-drug conjugate (ADC) of Formula I. In some embodiments,antibody-drug conjugates (ADC) can be prepared using a Linker havingreactive functionalities for covalently attaching to the drug and to theantibody. For example, in some embodiments, a cysteine thiol of anantibody (Ab) can form a bond with a reactive functional group of alinker or a drug-linker intermediate to make an ADC.

In one aspect, a linker has a functionality that is capable of reactingwith a free cysteine present on an antibody to form a covalent bond.Nonlimiting exemplary such reactive functionalities include maleimide,haloacetamides, α-haloacetyl, activated esters such as succinimideesters, 4-nitrophenyl esters, pentafluorophenyl esters,tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates, and isothiocyanates. See, e.g., the conjugationmethod at page 766 of Klussman, et al (2004), Bioconjugate Chemistry15(4):765-773, and the Examples herein.

In some embodiments, a linker has a functionality that is capable ofreacting with an electrophilic group present on an antibody. Exemplarysuch electrophilic groups include, but are not limited to, aldehyde andketone carbonyl groups. In some embodiments, a heteroatom of thereactive functionality of the linker can react with an electrophilicgroup on an antibody and form a covalent bond to an antibody unit.Nonlimiting exemplary such reactive functionalities include, but are notlimited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide.

A linker may comprise one or more linker components. Exemplary linkercomponents include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”),valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“MCC”). Various linker components are knownin the art, some of which are described below.

A linker may be a “cleavable linker,” facilitating release of a drug.Nonlimiting exemplary cleavable linkers include acid-labile linkers(e.g., comprising hydrazone), protease-sensitive (e.g.,peptidase-sensitive) linkers, photolabile linkers, ordisulfide-containing linkers (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020).

In certain embodiments, a linker has the following Formula II:

A_(a)-W_(w)-Y_(y)   Formula II

wherein A is a “stretcher unit”, and a is an integer from 0 to 1; W isan “amino acid unit”, and w is an integer from 0 to 12; Y is a “spacerunit”, and y is 0, 1, or 2; and Ab, D, and p are defined as above forFormula I. Exemplary embodiments of such linkers are described in U.S.Pat. No. 7,498,298, which is expressly incorporated herein by reference.

In some embodiments, a linker component comprises a “stretcher unit”that links an antibody to another linker component or to a drug moiety.Nonlimiting exemplary stretcher units are shown below (wherein the wavyline indicates sites of covalent attachment to an antibody, drug, oradditional linker components):

In some embodiments, a linker component comprises an “amino acid unit”.In some such embodiments, the amino acid unit allows for cleavage of thelinker by a protease, thereby facilitating release of the drug from theimmunoconjugate upon exposure to intracellular proteases, such aslysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784).Exemplary amino acid units include, but are not limited to, dipeptides,tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptidesinclude, but are not limited to, valine-citrulline (vc or val-cit),alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk orphe-lys); phenylalanine-homolysine (phe-homolys); andN-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include,but are not limited to, glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). An amino acid unit may compriseamino acid residues that occur naturally and/or minor amino acids and/ornon-naturally occurring amino acid analogs, such as citrulline Aminoacid units can be designed and optimized for enzymatic cleavage by aparticular enzyme, for example, a tumor-associated protease, cathepsinB, C and D, or a plasmin protease.

In some embodiments, a linker component comprises a “spacer” unit thatlinks the antibody to a drug moiety, either directly or through astretcher unit and/or an amino acid unit. A spacer unit may be“self-immolative” or a “non-self-immolative.” A “non-self-immolative”spacer unit is one in which part or all of the spacer unit remains boundto the drug moiety upon cleavage of the ADC. Examples ofnon-self-immolative spacer units include, but are not limited to, aglycine spacer unit and a glycine-glycine spacer unit. In someembodiments, enzymatic cleavage of an ADC containing a glycine-glycinespacer unit by a tumor-cell associated protease results in release of aglycine-glycine-drug moiety from the remainder of the ADC. In some suchembodiments, the glycine-glycine-drug moiety is subjected to ahydrolysis step in the tumor cell, thus cleaving the glycine-glycinespacer unit from the drug moiety.

A “self-immolative” spacer unit allows for release of the drug moiety.In certain embodiments, a spacer unit of a linker comprises ap-aminobenzyl unit. In some such embodiments, a p-aminobenzyl alcohol isattached to an amino acid unit via an amide bond, and a carbamate,methylcarbamate, or carbonate is made between the benzyl alcohol and thedrug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005)15:1087-1103). In some embodiments, the spacer unit isp-aminobenzyloxycarbonyl (PAB). In some embodiments, an ADC comprising aself-immolative linker has the structure:

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro, or-cyano; m is an integer ranging from 0 to 4; and p ranges from 1 toabout 20. In some embodiments, p ranges from 1 to 10, 1 to 7, 1 to 5, or1 to 4.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB group,such as 2-aminoimidazol-5-methanol derivatives (U.S. Pat. No. 7,375,078;Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- orpara-aminobenzylacetals. In some embodiments, spacers can be used thatundergo cyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides (Rodrigues et al (1995)Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (Storm et al (1972) J. Amer. Chem. Soc.94:5815) and 2-aminophenylpropionic acid amides (Amsberry, et al (1990)J. Org. Chem. 55:5867). Linkage of a drug to the α-carbon of a glycineresidue is another example of a self-immolative spacer that may beuseful in ADC (Kingsbury et al (1984) J. Med. Chem. 27:1447).

In some embodiments, linker L may be a dendritic type linker forcovalent attachment of more than one drug moiety to an antibody througha branching, multifunctional linker moiety (Sun et al (2002) Bioorganic& Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003) Bioorganic& Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase themolar ratio of drug to antibody, i.e. loading, which is related to thepotency of the ADC. Thus, where an antibody bears only one reactivecysteine thiol group, a multitude of drug moieties may be attachedthrough a dendritic linker.

Nonlimiting exemplary linkers are shown below in the context of an ADCof Formula I:

Further nonlimiting exemplary ADCs include the structures:

each R is independently H or C₁-C₆ alkyl; and n is 1 to 12.

Typically, peptide-type linkers can be prepared by forming a peptidebond between two or more amino acids and/or peptide fragments. Suchpeptide bonds can be prepared, for example, according to a liquid phasesynthesis method (e.g., E. Schröder and K. Lübke (1965) “The Peptides”,volume 1, pp 76-136, Academic Press).

In some embodiments, a linker is substituted with groups that modulatesolubility and/or reactivity. As a nonlimiting example, a chargedsubstituent such as sulfonate (—SO₃ ⁻) or ammonium may increase watersolubility of the linker reagent and facilitate the coupling reaction ofthe linker reagent with the antibody and/or the drug moiety, orfacilitate the coupling reaction of Ab-L (antibody-linker intermediate)with D, or D-L (drug-linker intermediate) with Ab, depending on thesynthetic route employed to prepare the ADC. In some embodiments, aportion of the linker is coupled to the antibody and a portion of thelinker is coupled to the drug, and then the Ab-(linker portion)^(a) iscoupled to drug-(linker portion)^(b) to form the ADC of Formula I. Insome such embodiments, the antibody comprises more than one (linkerportion)a substituents, such that more than one drug is coupled to theantibody in the ADC of Formula I.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with the following linker reagents:bis-malcincticio-trioxyethylene glycol (BMPEO),N-(β-maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS),N-(E-maleimidocaproyloxy) succinimide ester (EMCS),N-[γ-maleimidobutyryloxy]succinimide ester (GMBS),1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),4-(4-N-Maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA),succinimidyl (4-iodoacetyl)aminobenzoate (SIAB),N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl6-[(beta-maleimidopropionamido)hexanoate] (SMPH), iminothiolane (IT),sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and succinimidyl-(4-vinylsulfone)benzoate (SVSB), andincluding bis-maleimide reagents: dithiobismaleimidoethane (DTME),1,4-Bismaleimidobutane (BMB), 1,4 Bismaleimidyl-2,3-dihydroxybutane(BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)₂(shown below), and BM(PEG)₃ (shown below); bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCl), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In someembodiments, bis-maleimide reagents allow the attachment of the thiolgroup of a cysteine in the antibody to a thiol-containing drug moiety,linker, or linker-drug intermediate. Other functional groups that arereactive with thiol groups include, but are not limited to,iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyldisulfide, isocyanate, and isothiocyanate.

Certain useful linker reagents can be obtained from various commercialsources, such as Pierce Biotechnology, Inc. (Rockford, Ill.), MolecularBiosciences Inc. (Boulder, Colo.), or synthesized in accordance withprocedures described in the art; for example, in Toki et al (2002) J.Org. Chem. 67:1866-1872; Dubowchik, et al. (1997) Tetrahedron Letters,38:5257-60; Walker, M. A. (1995) J. Org. Chem. 60:5352-5355; Frisch etal (1996) Bioconjugate Chem. 7:180-186; U.S. Pat. No. 6,214,345; WO02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583; andWO 04/032828.

Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, e.g., WO94/11026.

b) Exemplary Drug Moieties

(1) Maytansine and Maytansinoids

In some embodiments, an immunoconjugate comprises an antibody conjugatedto one or more maytansinoid molecules. Maytansinoids are derivatives ofmaytansine, and are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinoids are disclosed, for example, in U.S. Pat. Nos.4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757;4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929;4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219;4,450,254; 4,362,663; and 4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody-drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification or derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through non-disulfide linkers to antibodies,(iii) stable in plasma, and (iv) effective against a variety of tumorcell lines.

Certain maytansinoids suitable for use as maytansinoid drug moieties areknown in the art and can be isolated from natural sources according toknown methods or produced using genetic engineering techniques (see,e.g., Yu et al (2002) PNAS 99:7968-7973). Maytansinoids may also beprepared synthetically according to known methods.

Exemplary maytansinoid drug moieties include, but are not limited to,those having a modified aromatic ring, such as: C-19-dechloro (U.S. Pat.No. 4,256,746) (prepared, for example, by lithium aluminum hydridereduction of ansamytocin P2); C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared, forexample, by demethylation using Streptomyces or Actinomyces ordechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (—OCOR),+/−dechloro (U.S. Pat. No. 4,294,757) (prepared, for example, byacylation using acyl chlorides), and those having modifications at otherpositions of the aromatic ring.

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared, forexample, by the reaction of maytansinol with H₂S or P₂S₅);C-14-alkoxymethyl(demethoxy/CH₂ OR) (U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared, for example, from Nocardia); C-15-hydroxy/acyloxy(U.S. Pat. No. 4,364,866) (prepared, for example, by the conversion ofmaytansinol by Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and4,315,929) (for example, isolated from Trewia nudlflora);C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared, forexample, by the demethylation of maytansinol by Streptomyces); and4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared, for example, by thetitanium trichloride/LAH reduction of maytansinol).

Many positions on maytansinoid compounds are useful as the linkageposition. For example, an ester linkage may be formed by reaction with ahydroxyl group using conventional coupling techniques. In someembodiments, the reaction may occur at the C-3 position having ahydroxyl group, the C-14 position modified with hydroxymethyl, the C-15position modified with a hydroxyl group, and the C-20 position having ahydroxyl group. In some embodiments, the linkage is formed at the C-3position of maytansinol or a maytansinol analogue.

Maytansinoid drug moieties include those having the structure:

where the wavy line indicates the covalent attachment of the sulfur atomof the maytansinoid drug moiety to a linker of an ADC. Each R mayindependently be H or a C₁-C₆ alkyl. The alkylene chain attaching theamide group to the sulfur atom may be methanyl, ethanyl, or propyl,i.e., m is 1, 2, or 3 (US 633410; U.S. Pat. No. 5,208,020; Chari et al(1992) Cancer Res. 52:127-131; Liu et al (1996) Proc. Natl. Acad. SciUSA 93:8618-8623).

All stereoisomers of the maytansinoid drug moiety are contemplated forthe ADC of the invention, i.e. any combination of R and S configurationsat the chiral carbons (U.S. Pat. No. 7,276,497; U.S. Pat. No. 6,913,748;U.S. Pat. No. 6,441,163; US 633410 (RE39151); U.S. Pat. No. 5,208,020;Widdison et al (2006) J. Med. Chem. 49:4392-4408, which are incorporatedby reference in their entirety). In some embodiments, the maytansinoiddrug moiety has the following stereochemistry:

Exemplary embodiments of maytansinoid drug moieties include, but are notlimited to, DM1; DM3; and DM4, having the structures:

wherein the wavy line indicates the covalent attachment of the sulfuratom of the drug to a linker (L) of an antibody-drug conjugate.

Other exemplary maytansinoid antibody-drug conjugates have the followingstructures and abbreviations (wherein Ab is antibody and p is 1 to about20. In some embodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is1 to 4):

Exemplary antibody-drug conjugates where DM1 is linked through a BMPEOlinker to a thiol group of the antibody have the structure andabbreviation:

where Ab is antibody; n is 0, 1, or 2; and p is 1 to about 20. In someembodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is 1 to 4.

Immunoconjugates containing maytansinoids, methods of making the same,and their therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020 and 5,416,064; US 2005/0276812 A1; and European Patent EP 0425 235 B1, the disclosures of which are hereby expressly incorporatedby reference. See also Liu et al. Proc. Natl. Acad. Sci. USA93:8618-8623 (1996); and Chari et al. Cancer Research 52:127-131 (1992).

In some embodiments, antibody-maytansinoid conjugates may be prepared bychemically linking an antibody to a maytansinoid molecule withoutsignificantly diminishing the biological activity of either the antibodyor the maytansinoid molecule. See, e.g., U.S. Pat. No. 5,208,020 (thedisclosure of which is hereby expressly incorporated by reference). Insome embodiments, ADC with an average of 3-4 maytansinoid moleculesconjugated per antibody molecule has shown efficacy in enhancingcytotoxicity of target cells without negatively affecting the functionor solubility of the antibody. In some instances, even one molecule oftoxin/antibody is expected to enhance cytotoxicity over the use of nakedantibody.

Exemplary linking groups for making antibody-maytansinoid conjugatesinclude, for example, those described herein and those disclosed in U.S.Pat. No. 5,208,020; EP Patent 0 425 235 B1; Chari et al. Cancer Research52:127-131 (1992); US 2005/0276812 A1; and US 2005/016993 A1, thedisclosures of which are hereby expressly incorporated by reference.

(2) Auristatins and Dolastatins

Drug moieties include dolastatins, auristatins, and analogs andderivatives thereof (U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588;U.S. Pat. No. 5,767,237; U.S. Pat. No. 6,124,431). Auristatins arederivatives of the marine mollusk compound dolastatin-10. While notintending to be bound by any particular theory, dolastatins andauristatins have been shown to interfere with microtubule dynamics, GTPhydrolysis, and nuclear and cellular division (Woyke et al (2001)Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer(U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998)Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin/auristatindrug moiety may be attached to the antibody through the N (amino)terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO02/088172; Doronina et al (2003) Nature Biotechnology 21(7):778-784;Francisco et al (2003) Blood 102(4):1458-1465).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties D_(E) and D_(F), disclosed in U.S.Pat. No. 7,498,298 and U.S. Pat. No. 7,659,241, the disclosures of whichare expressly incorporated by reference in their entirety:

wherein the wavy line of D_(E) and D_(F) indicates the covalentattachment site to an antibody or antibody-linker component, andindependently at each location:

R^(e) is selected from H and C₁-C₈ alkyl;

R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selectedfrom H, C₁-C₈ alkyl and C₃-C₈ carbocycle and n is selected from 2, 3, 4,5 and 6;

R⁶ is selected from H and C₁-C₈ alkyl;

R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈carbocycle and O—(C₁-C₈ alkyl);

R⁹is selected from H and C₁-C₈ alkyl;

R¹⁰ is selected from aryl or C₃-C₈ heterocycle;

Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;

R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,—(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₈ alkyl;

R¹⁴ is H or C₁-C₈ alkyl;

each occurrence of R¹⁵ is independently H, COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈ alkyl;

each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or—(CH₂)_(n)—COOH;

R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); and

n is an integer ranging from 0 to 6.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H or methyl. In an exemplary embodiment, R³ and R⁴are each isopropyl, R⁵ is —H, and R⁷ is sec-butyl.

In yet another embodiment, R² and R⁶ are each methyl, and R⁹ is —H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment, R¹⁰ is aryl.

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is —O—, R¹¹ is —H, methyl or t-butyl.

In one embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹³ is—(CH₂)_(n)—N(R¹⁶)₂, and R⁶ is —C₁-C₈ alkyl or —(CH₂)_(n)—COOH.

In another embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—SO₃H.

An exemplary auristatin embodiment of formula D_(E) is MMAE, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

An exemplary auristatin embodiment of formula D_(F) is MMAF, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

Other exemplary embodiments include monomethylvaline compounds havingphenylalanine carboxy modifications at the C-terminus of thepentapeptide auristatin drug moiety (WO 2007/008848) andmonomethylvaline compounds having phenylalanine sidechain modificationsat the C-terminus of the pentapeptide auristatin drug moiety (WO2007/008603).

Nonlimiting exemplary embodiments of ADC of Formula I comprising MMAE orMMAF and various linker components have the following structures andabbreviations (wherein “Ab” is an antibody; p is 1 to about 8, “Val-Cit”is a valine-citrulline dipeptide; and “S” is a sulfur atom:

Nonlimiting exemplary embodiments of ADCs of Formula I comprising MMAFand various linker components further include Ab-MC-PAB-MMAF andAb-PAB-MMAF Immunoconjugates comprising MMAF attached to an antibody bya linker that is not proteolytically cleavable have been shown topossess activity comparable to immunoconjugates comprising MMAF attachedto an antibody by a proteolytically cleavable linker (Doronina et al.(2006) Bioconjugate Chem. 17:114-124). In some such embodiments, drugrelease is believed to be effected by antibody degradation in the cell.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to a liquidphase synthesis method (see, e.g., E. Schröder and K. Lübke, “ThePeptides”, volume 1, pp 76-136, 1965, Academic Press).Auristatin/dolastatin drug moieties may, in some embodiments, beprepared according to the methods of: U.S. Pat. No. 7,498,298; U.S. Pat.No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem.Soc. 111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; Pettit et al(1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat.Biotechnol. 21(7):778-784.

In some embodiments, auristatin/dolastatin drug moieties of formulasD_(E) such as MMAE, and D_(F), such as MMAF, and drug-linkerintermediates and derivatives thereof, such as MC-MMAF, MC-MMAE,MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE, may be prepared using methodsdescribed in U.S. Pat. No. 7,498,298; Doronina et al. (2006)Bioconjugate Chem. 17:114-124; and Doronina et al. (2003) Nat. Biotech.21:778-784and then conjugated to an antibody of interest.

(3) Calicheamicin

In some embodiments, the immunoconjugate comprises an antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics, and analogues thereof, are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations (Hinman etal., (1993) Cancer Research 53:3336-3342; Lode et al., (1998) CancerResearch 58:2925-2928). Calicheamicin has intracellular sites of actionbut, in certain instances, does not readily cross the plasma membrane.Therefore, cellular uptake of these agents through antibody-mediatedinternalization may, in some embodiments, greatly enhances theircytotoxic effects. Nonlimiting exemplary methods of preparingantibody-drug conjugates with a calicheamicin drug moiety are described,for example, in U.S. Pat. No. 5,712,374; U.S. Pat. No. 5,714,586; U.S.Pat. No. 5,739,116; and U.S. Pat. No. 5,767,285.

(4) Pyrrolobenzodiazepines

In some embodiments, an ADC comprises a pyrrolobenzodiazepine (PBD). Insome embodiments, PDB dimers recognize and bind to specific DNAsequences. The natural product anthramycin, a PBD, was first reported in1965 (Leimgruber, et al., (1965) J. Am. Chem. Soc., 87:5793-5795;Leimgruber, et al., (1965) J. Am. Chem. Soc., 87:5791-5793). Since then,a number of PBDs, both naturally-occurring and analogues, have beenreported (Thurston, et al., (1994) Chem. Rev. 1994, 433-465 includingdimers of the tricyclic PBD scaffold (U.S. Pat. No. 6,884,799; U.S. Pat.No. 7,049,311; U.S. Pat. No. 7,067,511; U.S. Pat. No. 7,265,105; U.S.Pat. No. 7,511,032; U.S. Pat. No. 7,528,126; U.S. Pat. No. 7,557,099).Without intending to be bound by any particular theory, it is believedthat the dimer structure imparts the appropriate three-dimensional shapefor isohelicity with the minor groove of B-form DNA, leading to a snugfit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, NewYork, pp. 3-11 (1975); Hurley and Needham-VanDevanter, (1986) Acc. Chem.Res., 19:230-237). Dimeric PBD compounds bearing C2 aryl substituentshave been shown to be useful as cytotoxic agents (Hartley et al (2010)Cancer Res. 70(17):6849-6858; Antonow (2010) J. Med. Chem.53(7):2927-2941; Howard et al (2009) Bioorganic and Med. Chem. Letters19(22):6463-6466).

In some embodiments, PBD compounds can be employed as prodrugs byprotecting them at the N10 position with a nitrogen protecting groupwhich is removable in vivo (WO 00/12507; WO 2005/023814).

PBD dimers have been conjugated to antibodies and the resulting ADCshown to have anti-cancer properties (US 2010/0203007). Nonlimitingexemplary linkage sites on the PBD dimer include the five-memberedpyrrolo ring, the tether between the PBD units, and the N10-C11 iminegroup (WO 2009/016516; US 2009/304710; US 2010/047257; US 2009/036431;US 2011/0256157; WO 2011/130598).

Nonlimiting exemplary PBD dimer components of ADCs are of Formula A:

and salts and solvates thereof, wherein:

the wavy line indicates the covalent attachment site to the linker;

the dotted lines indicate the optional presence of a double bond betweenC1 and C2 or C2 and C3;

R² is independently selected from H, OH, ═O, ═CH₂, CN, R, OR, ═CH—R^(D),═C(R^(D))₂, O—SO₂—R, CO₂R and COR, and optionally further selected fromhalo or dihalo, wherein R^(D) is independently selected from R, CO₂R,COR, CHO, CO₂H, and halo;

R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,NHR, NRR′, NO₂, Me₃Sn and halo;

R⁷ is independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo;

Q is independently selected from O, S and NH;

R¹¹ is either H, or R or, where Q is O, SO₃M, where M is a metal cation;

R and R′ are each independently selected from optionally substitutedC₁₋₈ alkyl, C₁₋₁₂ alkyl, C₃₋₈ heterocyclyl, C₃₋₂₀ heterocycle, and C₅₋₂₀aryl groups, and optionally in relation to the group NRR′, R and R′together with the nitrogen atom to which they are attached form anoptionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring;

R¹², R¹⁶, R¹⁹ and R¹⁷ are as defined for R², R⁶, R⁹ and R⁷ respectively;

R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one ormore heteroatoms, e.g. O, S, N(H), NMe and/or aromatic rings, e.g.benzene or pyridine, which rings are optionally substituted; and

X and X′ are independently selected from O, S and N(H).

In some embodiments, R and R′ are each independently selected fromoptionally substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocycle, and C₅₋₂₀ arylgroups, and optionally in relation to the group NRR′, R and R′ togetherwith the nitrogen atom to which they are attached form an optionallysubstituted 4-, 5-, 6- or 7-membered heterocyclic ring.

In some embodiments, R⁹ and R¹⁹ are H.

In some embodiments, R⁶ and R¹⁶ are H.

In some embodiments, R⁷ are R¹⁷ are both OR^(7A), where R^(7A) isoptionally substituted C₁₋₄ alkyl. In some embodiments, R^(7A) is Me.

In some embodiments, X is O.

In some embodiments, R¹¹ is H.

In some embodiments, there is a double bond between C₂ and C₃ in eachmonomer unit.

In some embodiments, R² and R¹² are independently selected from H and R.In some embodiments, R² and R¹² are independently R. In someembodiments, R² and R¹² are independently optionally substituted C₅₋₂₀aryl or C₅₋₇ aryl or C₈₋₁₀ aryl. In some embodiments, R² and R¹² areindependently optionally substituted phenyl, thienyl, napthyl, pyridyl,quinolinyl, or isoquinolinyl. In some embodiments, R² and R¹² areindependently selected from ═O, ═CH₂, ═CH—R^(D), and ═C(R^(D))₂. In someembodiments, R² and R¹² are ═CH₂. In some embodiments, R² and R¹² areeach H. In some embodiments, R² and R¹² are each ═O. In someembodiments, R² and R¹² are each ═CF₂. In some embodiments, R² and/orR¹² are independently ═C(R^(D))₂. In some embodiments, R² and/or R¹² areindependently ═CH—R^(D).

In some embodiments, when R² and/or R¹² is ═CH—R^(D), each group mayindependently have either configuration shown below:

In some embodiments, a ═CH—R^(D) is in configuration (I).

In some embodiments, R″ is a C₃ alkylene group or a C₅ alkylene group.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(I):

wherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(II):

wherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(III):

wherein R^(E) and R^(E″) are each independently selected from H orR^(D), wherein R^(D) is defined as above; and

wherein n is 0 or 1.

In some embodiments, n is 0. In some embodiments, n is 1. In someembodiments, R^(E) and/or R^(E″) is H. In some embodiments, R^(E) andR^(E″) are H. In some embodiments, R^(E) and/or R^(E″) is R^(D), whereinR^(D) is optionally substituted C₁₋₁₂ alkyl. In some embodiments, R^(E)and/or R^(E″) is R^(D), wherein R^(D) is methyl.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(IV):

wherein Ar¹ and Ar² are each independently optionally substituted C₅₋₂₀aryl; wherein Ar¹ and Ar² may be the same or different; and

wherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(V):

wherein Ar¹ and Ar² are each independently optionally substituted C₅₋₂₀aryl; wherein Ar¹ and Ar² may be the same or different; and

wherein n is 0 or 1.

In some embodiments, Ar¹ and Ar² are each independently selected fromoptionally substituted phenyl, furanyl, thiophenyl and pyridyl. In someembodiments, Ar¹ and Ar² are each independently optionally substitutedphenyl. In some embodiments, Ar¹ and Ar² are each independentlyoptionally substituted thien-2-yl or thien-3-yl. In some embodiments,Ar¹ and Ar² are each independently optionally substituted quinolinyl orisoquinolinyl. The quinolinyl or isoquinolinyl group may be bound to thePBD core through any available ring position. For example, thequinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4y1,quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. In someembodiments, the quinolinyl is selected from quinolin-3-yl andquinolin-6-yl. The isoquinolinyl may be isoquinolin-1-yl,isoquinolin-3-yl, isoquinolin-4y1, isoquinolin-5-yl, isoquinolin-6-yl,isoquinolin-7-yl and isoquinolin-8-yl. In some embodiments, theisoquinolinyl is selected from isoquinolin-3-yl and isoquinolin-6-yl.

Further nonlimiting exemplary PBD dimer components of ADCs are ofFormula B:

and salts and solvates thereof, wherein:

the wavy line indicates the covalent attachment site to the linker;

the wavy line connected to the OH indicates the S or R configuration;

R^(V1) and R^(V2) are independently selected from H, methyl, ethyl andphenyl (which phenyl may be optionally substituted with fluoro,particularly in the 4 position) and C₅-6 heterocyclyl; and

n is 0 or 1.

In some embodiments, R^(V1) and R^(V2) are independently selected fromH, phenyl, and 4-fluorophenyl.

In some embodiments, a linker may be attached at one of various sites ofthe PBD dimer drug moiety, including the N10 imine of the B ring, theC-2 endo/exo position of the C ring, or the tether unit linking the Arings (see structures C(I) and C(II) below).

Nonlimiting exemplary PBD dimer components of ADCs include Formulas C(I)and C(II):

Formulas C(I) and C(II) are shown in their N10-C11 imine form. ExemplaryPBD drug moieties also include the carbinolamine and protectedcarbinolamine forms as well, as shown in the box below:

wherein:

X is CH₂ (n=1 to 5), N, or O;

Z and Z′ are independently selected from OR and NR₂, where R is aprimary, secondary or tertiary alkyl chain containing 1 to 5 carbonatoms;

R₁, R′₁, R₂ and R′₂ are each independently selected from H, C₁-C₈ alkyl,C₂-C₈ alkenyl, C₂C₈ alkynyl, C₅₋₂₀ aryl (including substituted aryls),C₅₋₂₀ heteroaryl groups, —NH₂, —NHMe, —OH, and —SH, where, in someembodiments, alkyl, alkenyl and alkynyl chains comprise up to 5 carbonatoms;

R₃ and R′₃ are independently selected from H, OR, NHR, and NR₂, where Ris a primary, secondary or tertiary alkyl chain containing 1 to 5 carbonatoms;

R₄ and R′₄ are independently selected from H, Me, and OMe;

R₅ is selected from C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₅₋₂₀aryl (including aryls substituted by halo, nitro, cyano, alkoxy, alkyl,heterocyclyl) and C₅₋₂₀ heteroaryl groups, where, in some embodiments,alkyl, alkenyl and alkynyl chains comprise up to 5 carbon atoms;

R₁₁ is H, C₁-C₈ alkyl, or a protecting group (such as acetyl,trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ),9-fluorenylmethylenoxycarbonyl (Fmoc), or a moiety comprising aself-immolating unit such as valine-citrulline-PAB);

R₁₂ is is H, C₁-C₈ alkyl, or a protecting group;

wherein a hydrogen of one of R₁, R′₁, R₂, R′₂, R₅, or R₁₂ or a hydrogenof the —OCH₂CH₂(X)_(n)CH₂CH₂O— spacer between the A rings is replacedwith a bond connected to the linker of the ADC.

Exemplary PDB dimer portions of ADC include, but are not limited to (thewavy line indicates the site of covalent attachment to the linker):

Nonlimiting exemplary embodiments of ADCs comprising PBD dimers have thefollowing structures:

-   -   PBD dimer-Phe-Lys-PAB-Ab, wherein:

n is 0 to 12. In some embodiments, n is 2 to 10. In some embodiments, nis 4 to 8. In some embodiments, n is selected from 4 and 8.

The linkers of PBD dimer-val-cit-PAB-Ab and the PBD dimer-Phe-Lys-PAB-Abare protease cleavable, while the linker of PBD dimer-maleimide-acetalis acid-labile.

PBD dimers and ADC comprising PBD dimers may be prepared according tomethods known in the art. See, e.g., WO 2009/016516; US 2009/304710; US2010/047257; US 2009/036431; US 2011/0256157; WO 2011/130598.

(5) Anthracyclines

In some embodiments, an ADC comprising anthracycline. Anthracyclines areantibiotic compounds that exhibit cytotoxic activity. While notintending to be bound by any particular theory, studies have indicatedthat anthracyclines may operate to kill cells by a number of differentmechanisms, including: 1) intercalation of the drug molecules into theDNA of the cell thereby inhibiting DNA-dependent nucleic acid synthesis;2) production by the drug of free radicals which then react withcellular macromolecules to cause damage to the cells, and/or 3)interactions of the drug molecules with the cell membrane (see, e.g., C.Peterson et al., “Transport And Storage Of Anthracycline In ExperimentalSystems And Human Leukemia” in Anthracycline Antibiotics In CancerTherapy; N. R. Bachur, “Free Radical Damage” id. at pp.97-102). Becauseof their cytotoxic potential anthracyclines have been used in thetreatment of numerous cancers such as leukemia, breast carcinoma, lungcarcinoma, ovarian adenocarcinoma and sarcomas (see e.g., P. H-Wiernik,in Anthracycline: Current Status And New Developments p 11).

Nonlimiting exemplary anthracyclines include doxorubicin, epirubicin,idarubicin, daunomycin, nemorubicin, and derivatives thereof.Immunoconjugates and prodrugs of daunorubicin and doxorubicin have beenprepared and studied (Kratz et al (2006) Current Med. Chem. 13:477-523;Jeffrey et al (2006) Bioorganic & Med. Chem. Letters 16:358-362; Torgovet al (2005) Bioconj. Chem. 16:717-721; Nagy et al (2000) Proc. Natl.Acad. Sci. USA 97:829-834; Dubowchik et al (2002) Bioorg. & Med. Chem.Letters 12:1529-1532; King et al (2002) J. Med. Chem. 45:4336-4343; EP0328147; U.S. Pat. No. 6,630,579). The antibody-drug conjugateBR96-doxorubicin reacts specifically with the tumor-associated antigenLewis-Y and has been evaluated in phase I and II studies (Saleh et al(2000) J. Clin. Oncology 18:2282-2292; Ajani et al (2000) Cancer Jour.6:78-81; Tolcher et al (1999) J. Clin. Oncology 17:478-484).

PNU-159682 is a potent metabolite (or derivative) of nemorubicin(Quintieri, et al. (2005) Clinical Cancer Research 11(4):1608-1617).Nemorubicin is a semisynthetic analog of doxorubicin with a2-methoxymorpholino group on the glycoside amino of doxorubicin and hasbeen under clinical evaluation (Grandi et al (1990) Cancer Treat. Rev.17:133; Ripamonti et al (1992) Brit. J. Cancer 65:703;), including phaseII/III trials for hepatocellular carcinoma (Sun et al (2003) Proceedingsof the American Society for Clinical Oncology 22, Abs1448; Quintieri(2003) Proceedings of the American Association of Cancer Research,44:1st Ed, Abs 4649; Pacciarini et al (2006) Jour. Clin. Oncology24:14116).

A nonlimiting exemplary ADC comprising nemorubicin or nemorubicinderivatives is shown in Formula Ia:

wherein R₁ is hydrogen atom, hydroxy or methoxy group and R₂ is a C₁-C₅alkoxy group, or a pharmaceutically acceptable salt thereof;

L₁ and Z together are a linker (L) as described herein;

T is an antibody (Ab) as described herein; and

m is 1 to about 20. In some embodiments, m is 1 to 10, 1 to 7, 1 to 5,or 1 to 4.

In some embodiments, R₁ and R₂ are both methoxy (—OMe).

A further nonlimiting exemplary ADC comprising nemorubicin ornemorubicin derivatives is shown in Formula Ib:

wherein R₁ is hydrogen atom, hydroxy or methoxy group and R₂ is a C₁-C₅alkoxy group, or a pharmaceutically acceptable salt thereof;

L₂ and Z together are a linker (L) as described herein;

T is an antibody (Ab) as described herein; and

m is 1 to about 20. In some embodiments, m is 1 to 10, 1 to 7, 1 to 5,or 1 to 4.

In some embodiments, R₁ and R₂ are both methoxy (—OMe).

In some embodiments, the nemorubicin component of anemorubicin-containing ADC is PNU-159682. In some such embodiments, thedrug portion of the ADC may have one of the following structures:

wherein the wavy line indicates the attachment to the linker (L).

Anthracyclines, including PNU-159682, may be conjugated to antibodiesthrough several linkage sites and a variety of linkers (US 2011/0076287;WO2009/099741; US 2010/0034837; WO 2010/009124) , including the linkersdescribed herein.

Exemplary ADCs comprising a nemorubicin and linker include, but are notlimited to:

wherein: R₁ and R₂ are independently selected from H and C₁-C₆ alkyl;and

The linker of PNU-159682 maleimide acetal-Ab is acid-labile, while thelinkers of PNU-159682-val-cit-PAB-Ab, PNU-159682-val-cit-PAB-spacer-Ab,and PNU-159682-val-cit-PAB-spacer(R¹R²)-Ab are protease cleavable.

(6) Other Drug Moieties

Drug moieties also include geldanamycin (Mandler et al (2000) J. Nat.Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791); and enzymatically active toxins and fragments thereof,including, but not limited to, diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.See, e.g., WO 93/21232.

Drug moieties also include compounds with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease).

In certain embodiments, an immunoconjugate may comprise a highlyradioactive atom. A variety of radioactive isotopes are available forthe production of radioconjugated antibodies. Examples include At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactiveisotopes of Lu. In some embodiments, when an immunoconjugate is used fordetection, it may comprise a radioactive atom for scintigraphic studies,for example Tc⁹⁹ or I¹²³, or a spin label for nuclear magnetic resonance(NMR) imaging (also known as magnetic resonance imaging, MRI), such aszirconium-89, iodine-123, iodine-131, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.Zirconium-89 may be complexed to various metal chelating agents andconjugated to antibodies, e.g., for PET imaging (WO 2011/056983).

The radio- or other labels may be incorporated in the immunoconjugate inknown ways. For example, a peptide may be biosynthesized or chemicallysynthesized using suitable amino acid precursors comprising, forexample, one or more fluorine-19 atoms in place of one or morehydrogens. In some embodiments, labels such as Tc⁹⁹, I¹²³, Re¹⁸⁶, Re¹⁸⁸and In¹¹¹ can be attached via a cysteine residue in the antibody. Insome embodiments, yttrium-90 can be attached via a lysine residue of theantibody. In some embodiments, the IODOGEN method (Fraker et al (1978)Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporateiodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRCPress 1989) describes certain other methods.

In certain embodiments, an immunoconjugate may comprise an antibodyconjugated to a prodrug-activating enzyme. In some such embodiments, aprodrug-activating enzyme converts a prodrug (e.g., a peptidylchemotherapeutic agent, see WO 81/01145) to an active drug, such as ananti-cancer drug. Such immunoconjugates are useful, in some embodiments,in antibody-dependent enzyme-mediated prodrug therapy (“ADEPT”). Enzymesthat may be conjugated to an antibody include, but are not limited to,alkaline phosphatases, which are useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatases, which areuseful for converting sulfate-containing prodrugs into free drugs;cytosine deaminase, which is useful for converting non-toxic5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases,such as serratia protease, thermolysin, subtilisin, carboxypeptidasesand cathepsins (such as cathepsins B and L), which are useful forconverting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, which are useful for converting prodrugs thatcontain D-amino acid substituents; carbohydrate-cleaving enzymes such asβ-galactosidase and neuraminidase, which are useful for convertingglycosylated prodrugs into free drugs; β-lactamase, which is useful forconverting drugs derivatized with β-lactams into free drugs; andpenicillin amidases, such as penicillin V amidase and penicillin Gamidase, which are useful for converting drugs derivatized at theiramine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,into free drugs. In some embodiments, enzymes may be covalently bound toantibodies by recombinant DNA techniques well known in the art. See,e.g., Neuberger et al., Nature 312:604-608 (1984).

c) Drug Loading

Drug loading is represented by p, the average number of drug moietiesper antibody in a molecule of Formula I. Drug loading may range from 1to 20 drug moieties (D) per antibody. ADCs of Formula I includecollections of antibodies conjugated with a range of drug moieties, from1 to 20. The average number of drug moieties per antibody inpreparations of ADC from conjugation reactions may be characterized byconventional means such as mass spectroscopy, ELISA assay, and HPLC. Thequantitative distribution of ADC in terms of p may also be determined.In some instances, separation, purification, and characterization ofhomogeneous ADC where p is a certain value from ADC with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in certain exemplary embodiments above, an antibodymay have only one or several cysteine thiol groups, or may have only oneor several sufficiently reactive thiol groups through which a linker maybe attached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the average drug loading for an ADC ranges from 1 to about8; from about 2 to about 6; or from about 3 to about 5. Indeed, it hasbeen shown that for certain ADCs, the optimal ratio of drug moieties perantibody may be less than 8, and may be about 2 to about 5 (U.S. Pat.No. 7,498,298).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, and for example, by: (i) limiting the molar excess ofdrug-linker intermediate or linker reagent relative to antibody, (ii)limiting the conjugation reaction time or temperature, and (iii) partialor limiting reductive conditions for cysteine thiol modification.

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent, then theresulting product is a mixture of ADC compounds with a distribution ofone or more drug moieties attached to an antibody. The average number ofdrugs per antibody may be calculated from the mixture by a dual ELISAantibody assay, which is specific for antibody and specific for thedrug. Individual ADC molecules may be identified in the mixture by massspectroscopy and separated by HPLC, e.g. hydrophobic interactionchromatography (see, e.g., McDonagh et al (2006) Prot. Engr. Design &Selection 19(7):299-307; Hamblett et al (2004) Clin. Cancer Res.10:7063-7070; Hamblett, K. J., et al. “Effect of drug loading on thepharmacology, pharmacokinetics, and toxicity of an anti-CD30antibody-drug conjugate,” Abstract No. 624, American Association forCancer Research, 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings ofthe AACR, Volume 45, March 2004; Alley, S. C., et al. “Controlling thelocation of drug attachment in antibody-drug conjugates,” Abstract No.627, American Association for Cancer Research, 2004 Annual Meeting, Mar.27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certainembodiments, a homogeneous ADC with a single loading value may beisolated from the conjugation mixture by electrophoresis orchromatography.

d) Certain Methods of Preparing Immunoconiugates

An ADC of Formula I may be prepared by several routes employing organicchemistry reactions, conditions, and reagents known to those skilled inthe art, including: (1) reaction of a nucleophilic group of an antibodywith a bivalent linker reagent to form Ab-L via a covalent bond,followed by reaction with a drug moiety D; and (2) reaction of anucleophilic group of a drug moiety with a bivalent linker reagent, toform D-L, via a covalent bond, followed by reaction with a nucleophilicgroup of an antibody. Exemplary methods for preparing an ADC of FormulaI via the latter route are described in U.S. Pat. No. 7,498,298, whichis expressly incorporated herein by reference.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; and (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol) or tricarbonylethylphosphine (TCEP), such that theantibody is fully or partially reduced. Each cysteine bridge will thusform, theoretically, two reactive thiol nucleophiles. Additionalnucleophilic groups can be introduced into antibodies throughmodification of lysine residues, e.g., by reacting lysine residues with2-iminothiolane (Traut's reagent), resulting in conversion of an amineinto a thiol. Reactive thiol groups may also be introduced into anantibody by introducing one, two, three, four, or more cysteine residues(e.g., by preparing variant antibodies comprising one or more non-nativecysteine amino acid residues).

Antibody-drug conjugates of the invention may also be produced byreaction between an electrophilic group on an antibody, such as analdehyde or ketone carbonyl group, with a nucleophilic group on a linkerreagent or drug. Useful nucleophilic groups on a linker reagent include,but are not limited to, hydrazide, oxime, amino, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In oneembodiment, an antibody is modified to introduce electrophilic moietiesthat are capable of reacting with nucleophilic substituents on thelinker reagent or drug. In another embodiment, the sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither galactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the antibody that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, antibodies containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Such analdehyde can be reacted with a drug moiety or linker nucleophile.

Exemplary nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Nonlimiting exemplary cross-linker reagents that may be used to prepareADC are described herein in the section titled “Exemplary Linkers.”Methods of using such cross-linker reagents to link two moieties,including a proteinaceous moiety and a chemical moiety, are known in theart. In some embodiments, a fusion protein comprising an antibody and acytotoxic agent may be made, e.g., by recombinant techniques or peptidesynthesis. A recombinant DNA molecule may comprise regions encoding theantibody and cytotoxic portions of the conjugate either adjacent to oneanother or separated by a region encoding a linker peptide which doesnot destroy the desired properties of the conjugate.

In yet another embodiment, an antibody may be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pre-targeting whereinthe antibody-receptor conjugate is administered to the patient, followedby removal of unbound conjugate from the circulation using a clearingagent and then administration of a “ligand” (e.g., avidin) which isconjugated to a cytotoxic agent (e.g., a drug or radionucleotide).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-LY6E antibodies provided hereinis useful for detecting the presence of LY6E in a biological sample. Theterm “detecting” as used herein encompasses quantitative or qualitativedetection. A “biological sample” comprises, e.g., a cell or tissue(e.g., biopsy material, including cancerous or potentially cancerouscolon, colorectal, endometrial, pancreatic, or ovarian tissue).

In one embodiment, an anti-LY6E antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of LY6E in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-LY6E antibody as described herein under conditionspermissive for binding of the anti-LY6E antibody to LY6E, and detectingwhether a complex is formed between the anti-LY6E antibody and LY6E inthe biological sample. Such method may be an in vitro or in vivo method.In one embodiment, an anti-LY6E antibody is used to select subjectseligible for therapy with an anti-LY6E antibody, e.g. where LY6E is abiomarker for selection of patients. In a further embodiment, thebiological sample is a cell or tissue (e.g., biopsy material, includingcancerous or potentially cancerous colon, colorectal, endometrial,pancreatic, or ovarian tissue).

In a further embodiment, an anti-LY6E antibody is used in vivo todetect, e.g., by in vivo imaging, A LY6E-positive cancer in a subject,e.g., for the purposes of diagnosing, prognosing, or staging cancer,determining the appropriate course of therapy, or monitoring response ofa cancer to therapy. One method known in the art for in vivo detectionis immuno-positron emission tomography (immuno-PET), as described, e.g.,in van Dongen et al., The Oncologist 12:1379-1389 (2007) and Verel etal., J. Nucl. Med. 44:1271-1281 (2003). In such embodiments, a method isprovided for detecting A LY6E-positive cancer in a subject, the methodcomprising administering a labeled anti-LY6E antibody to a subjecthaving or suspected of having A LY6E-positive cancer, and detecting thelabeled anti-LY6E antibody in the subject, wherein detection of thelabeled anti-LY6E antibody indicates A LY6E-positive cancer in thesubject. In certain of such embodiments, the labeled anti-LY6E antibodycomprises an anti-LY6E antibody conjugated to a positron emitter, suchas ⁶⁸Ga, ¹⁸F, ⁶⁴Cu, ⁸⁶Y, ⁷⁶Br, ⁸⁹Zr, and ¹²⁴I. In a particularembodiment, the positron emitter is ⁸⁹Zr.

In further embodiments, a method of diagnosis or detection comprisescontacting a first anti-LY6E antibody immobilized to a substrate with abiological sample to be tested for the presence of LY6E, exposing thesubstrate to a second anti-LY6E antibody, and detecting whether thesecond anti-LY6E is bound to a complex between the first anti-LY6Eantibody and LY6E in the biological sample. A substrate may be anysupportive medium, e.g., glass, metal, ceramic, polymeric beads, slides,chips, and other substrates. In certain embodiments, a biological samplecomprises a cell or tissue (e.g., biopsy material, including cancerousor potentially cancerous colorectal, endometrial, pancreatic or ovariantissue). In certain embodiments, the first or second anti-LY6E antibodyis any of the antibodies described herein. In such embodiments, thesecond anti-LY6E antibody may be 6D3 or 7C9; or antibodies derived from6D3 or 7C9 as described herein.

Exemplary disorders that may be diagnosed or detected according to anyof the above embodiments include LY6E-positive cancers, such asLY6E-positive colorectal cancer (including adenocarcinoma),LY6E-positive ovarian cancer (including ovarian serous adenocarcinoma),LY6E-positive pancreatic cancer (including pancreatic ductaladenocarcinoma), and LY6E-positive endometrial cancer. In someembodiments, A LY6E-positive cancer is a cancer that receives ananti-LY6E immunohistochemistry (IHC) or in situ hybridization (ISH)score greater than “0,” which corresponds to very weak or no stainingin >90% of tumor cells, under the conditions described herein in ExampleB. In another embodiment, A LY6E-positive cancer expresses LY6E at a 1+,2+or 3+ level, as defined under the conditions described herein inExample B. In some embodiments, A LY6E-positive cancer is a cancer thatexpresses LY6E according to a reverse-transcriptase PCR (RT-PCR) assaythat detects LY6E mRNA. In some embodiments, the RT-PCR is quantitativeRT-PCR.

In certain embodiments, labeled anti-LY6E antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like. In anotherembodiment, a label is a positron emitter. Positron emitters include butare not limited to ⁶⁸Ga, ¹⁸F, ⁶⁴Cu, ⁸⁶Y, ⁷⁶Br, ⁸⁹Zr, and ¹²⁴I. In aparticular embodiment, a positron emitter is ⁸⁹Zr.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-LY6E antibody or immunoconjugateas described herein are prepared by mixing such antibody orimmunoconjugate having the desired degree of purity with one or moreoptional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody or immunoconjugate formulations aredescribed in U.S. Pat. No. 6,267,958. Aqueous antibody orimmunoconjugate formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations including ahistidine-acetate buffer.

The formulation herein may also contain more than one active ingredientas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, in some instances, it may be desirable to furtherprovide a platinum complex, e.g., for the treatment of LY6E-positivecancer such as, for example, a LY6E-positive breast cancer, or aLY6E-positive pancreatic cancer, or a LY6E-positive colon cancer, or aLY6E-positive colorectal cancer, or a LY6E-positive melanoma cancer, ora LY6E-positive ovarian cancer, or a LY6E-positive non-small cell lungcancer, or a LY6E-positive gastric cancer.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody or immunoconjugate, whichmatrices are in the form of shaped articles, e.g. films, ormicrocapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-LY6E antibodies or immunoconjugates provided herein maybe used in methods, e.g., therapeutic methods.

In one aspect, an anti-LY6E antibody or immunoconjugate provided hereinis used in a method of inhibiting proliferation of a LY6E-positive cell,the method comprising exposing the cell to the anti-LY6E antibody orimmunoconjugate under conditions permissive for binding of the anti-LY6Eantibody or immunoconjugate to LY6E on the surface of the cell, therebyinhibiting the proliferation of the cell. In certain embodiments, themethod is an in vitro or an in vivo method. In further embodiments, thecell is a breast cancer cell or a pancreatic cancer cell or a coloncancer cell, or a colorectal cancer cell, or a melanoma cancer cell, oran ovarian cancer cell, or a non-small cell lung cancer cell, or agastric cancer cell.

Inhibition of cell proliferation in vitro may be assayed using theCellTiter-Glo™ Luminescent Cell Viability Assay, which is commerciallyavailable from Promega (Madison, Wis.). That assay determines the numberof viable cells in culture based on quantitation of ATP present, whichis an indication of metabolically active cells. See Crouch et al. (1993)J. Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677. The assay may beconducted in 96- or 384-well format, making it amenable to automatedhigh-throughput screening (HTS). See Cree et al. (1995) AntiCancer Drugs6:398-404. The assay procedure involves adding a single reagent(CellTiter-Glo® Reagent) directly to cultured cells. This results incell lysis and generation of a luminescent signal produced by aluciferase reaction. The luminescent signal is proportional to theamount of ATP present, which is directly proportional to the number ofviable cells present in culture. Data can be recorded by luminometer orCCD camera imaging device. The luminescence output is expressed asrelative light units (RLU).

In another aspect, an anti-LY6E antibody or immunoconjugate for use as amedicament is provided. In further aspects, an anti-LY6E antibody orimmunoconjugate for use in a method of treatment is provided. In certainembodiments, an anti-LY6E antibody or immunoconjugate for use intreating LY6E-positive cancer is provided. In certain embodiments, theinvention provides an anti-LY6E antibody or immunoconjugate for use in amethod of treating an individual having a LY6E-positive cancer, themethod comprising administering to the individual an effective amount ofthe anti-LY6E antibody or immunoconjugate. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, e.g., as describedbelow.

In a further aspect, the invention provides for the use of an anti-LY6Eantibody or immunoconjugate in the manufacture or preparation of amedicament. In one embodiment, the medicament is for treatment ofLY6E-positive cancer. In a further embodiment, the medicament is for usein a method of treating LY6E-positive cancer, the method comprisingadministering to an individual having LY6E-positive cancer an effectiveamount of the medicament. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, e.g., as described below.

In a further aspect, the invention provides a method for treatingLY6E-positive cancer. In one embodiment, the method comprisesadministering to an individual having such LY6E-positive cancer aneffective amount of an anti-LY6E antibody or immunoconjugate. In onesuch embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, as described below.

A LY6E-positive cancer according to any of the above embodiments may be,e.g., LY6E-positive breast cancer, or LY6E-positive pancreatic cancer,or LY6E-positive colon cancer, or LY6E-positive colorectal cancer, orLY6E-positive melanoma cancer, or LY6E-positive ovarian cancer, orLY6E-positive non-small cell lung cancer, or LY6E-positive gastriccancer. In some embodiments, a LY6E-positive cancer is a cancer thatreceives an anti-LY6E immunohistochemistry (IHC) or in situhybridization (ISH) score greater than “0,” which corresponds to veryweak or no staining in >90% of tumor cells, under the conditionsdescribed herein. In another embodiment, a LY6E-positive cancerexpresses LY6E at a 1+, 2+or 3+ level, as defined under the conditionsdescribed herein. In some embodiments, a LY6E-positive cancer is acancer that expresses LY6E according to a reverse-transcriptase PCR(RT-PCR) assay that detects LY6E mRNA. In some embodiments, the RT-PCRis quantitative RT-PCR.

An “individual” according to any of the above embodiments may be ahuman.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-LY6E antibodies or immunoconjugate providedherein, e.g., for use in any of the above therapeutic methods. In oneembodiment, a pharmaceutical formulation comprises any of the anti-LY6Eantibodies or immunoconjugates provided herein and a pharmaceuticallyacceptable carrier. In another embodiment, a pharmaceutical formulationcomprises any of the anti-LY6E antibodies or immunoconjugates providedherein and at least one additional therapeutic agent, e.g., as describedbelow.

Antibodies or immunoconjugates of the invention can be used either aloneor in combination with other agents in a therapy. For instance, anantibody or immunoconjugate of the invention may be co-administered withat least one additional therapeutic agent. In certain embodiments, anadditional therapeutic agent is a platinum complex, e.g., for thetreatment of LY6E-positive cancer such as, for example, a LY6E-positivebreast cancer, or a LY6E-positive pancreatic cancer, or a LY6E-positivecolon cancer, or a LY6E-positive colorectal cancer, or a LY6E-positivemelanoma cancer, or a LY6E-positive ovarian cancer, or a LY6E-positivenon-small cell lung cancer, or a LY6E-positive gastric cancer.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody or immunoconjugate of the invention canoccur prior to, simultaneously, and/or following, administration of theadditional therapeutic agent and/or adjuvant. Antibodies orimmunoconjugates of the invention can also be used in combination withradiation therapy.

An antibody or immunoconjugate of the invention (and any additionaltherapeutic agent) can be administered by any suitable means, includingparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies or immunoconjugates of the invention would be formulated,dosed, and administered in a fashion consistent with good medicalpractice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The antibody or immunoconjugate need notbe, but is optionally formulated with one or more agents currently usedto prevent or treat the disorder in question. The effective amount ofsuch other agents depends on the amount of antibody or immunoconjugatepresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody or immunoconjugate of the invention (when used alone or incombination with one or more other additional therapeutic agents) willdepend on the type of disease to be treated, the type of antibody orimmunoconjugate, the severity and course of the disease, whether theantibody or immunoconjugate is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody or immunoconjugate, and the discretion ofthe attending physician. The antibody or immunoconjugate is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody or immunoconjugate can be aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. One typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs. One exemplary dosage of the antibody orimmunoconjugate would be in the range from about 0.05 mg/kg to about 10mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kgor 10 mg/kg (or any combination thereof) may be administered to thepatient. Such doses may be administered intermittently, e.g. every weekor every three weeks (e.g. such that the patient receives from about twoto about twenty, or e.g. about six doses of the antibody). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using both an immunoconjugate of theinvention and an anti-LY6E antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thedisorder and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody or immunoconjugate of the invention. Thelabel or package insert indicates that the composition is used fortreating the condition of choice. Moreover, the article of manufacturemay comprise (a) a first container with a composition contained therein,wherein the composition comprises an antibody or immunoconjugate of theinvention; and (b) a second container with a composition containedtherein, wherein the composition comprises a further cytotoxic orotherwise therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition. Alternatively, or additionally, the article of manufacturemay further comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution ordextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

I. Sequences of the Invention

In another aspect of the invention, the following sequences useful forthe treatment, prevention and/or diagnosis of the disorders describedabove are provided.

SEQ ID NO: SEQUENCE DESCRIPTION 1 MKIFLPVLLAALLGVERASSLMCFSCLNQKSNHUMAN Ly6E amino acid LYCLKPTICSDQDNYCVTVSASAGIGNLVTFGsequence with signal sequence HSLSKTCSPACPIPEGVNVGVASMGISCCQSFL(amino acids 1-20, underlined) CNFSAADGGLRASVTLLGAGLLLSLLPALLRFGP 2MKIFLPVLLAALLGVERASSLMCFSCLNQKSN CYNOMOLOGOUS Ly6ELYCLKPTICSDQDNYCVTVSTSAGIGNLVTFG amino acid sequence withHSLSKTCSPACPLPEGINVGVASMGISCCQSFL signal sequence (amino acidsCNFSAADGGLRASATLLGAGLLLSLLPALLRFGP 1-20, underlined) 3DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNW Humanized variable light chainYQQKPGKTVKLLIYYTSNLHSGVPSRFSGSGSGT amino acid sequence of anti-DYTLTISSLQPEDFATYYCQQYSELPWTFGQGTK Ly6E antibody clone hu9B12 VEIK v12 4DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNW Chimeric variable light chainYQQKPDGTVKLLIYYTSNLHSGVPSRFSGSGSGT amino acid sequence of anti-DYSLTISNLEPEDIATYYCQQYSELPWTFGGGTK Ly6E antibody clone xLy6E VEIK mu9B125 EVQLVESGPALVKPTQTLTLTCTVSGFSLTGYSVN Humanized variable heavyWIRQPPGKALEWLGMIWGDGSTDYNSALKSRLTI chain amino acid sequence ofSKDTSKNQVVLTMTNMDPVDTATYYCARDYYFN anti-Ly6E antibody cloneYASWFAYWGQGTLVTVSS hu9B12 v12 6 QVQLKESGPGLVAPSQSLSLTCTVSGFSLTGYSVNChimeric variable heavy chain WVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLamino acid sequence of anti- TISKDNSKSQVFLKMNSLQTDDTARYYCARDYYLy6E antibody clone xLy6E FNYASWFAYWGPGTLVTVSA mu9B12 7 SASQGISNYLNHumanized variable light chain CDR 1 amino acid sequence ofanti-Ly6E antibody clone hu9B12 v12 8 YTSNLHSHumanized variable light chain CDR 2 amino acid sequence ofanti-Ly6E antibody clone hu9B12 v12 9 QQYSELPWTHumanized variable light chain CDR 3 amino acid sequence ofanti-Ly6E antibody clone hu9B12 v12 10 GFSLTGYSVNHumanized variable heavy chain CDR 1 amino acid sequence of anti-Ly6Eantibody clone hu9B12 v12 11 MIWGDGSTDYNSALKS Humanized variable heavychain CDR 2 amino acid sequence of anti-Ly6E antibody clone hu9B12 v1212 DYYFNYASWFAY Humanized variable heavy chain CDR 3 amino acidsequence of anti-Ly6E antibody clone hu9B12 v12 13 SASQGISNYLNChimeric variable light chain CDR 1 amino acid sequence ofanti-Ly6E antibody clone xLy6E mu9B12 14 YTSNLHSChimeric variable light chain CDR 2 amino acid sequence ofanti-Ly6E antibody clone xLy6E mu9B12 15 QQYSELPWTChimeric variable light chain CDR 3 amino acid sequence ofanti-Ly6E antibody clone xLy6E mu9B12 16 GFSLTGYSVNChimeric variable heavy chain CDR 1 amino acid sequence ofanti-Ly6E antibody clone xLy6E mu9B12 17 MIWGDGSTDYNSALKSChimeric variable heavy chain CDR 2 amino acid sequence ofanti-Ly6E antibody clone xLy6E mu9B12 18 DYYFNYASWFAYChimeric variable heavy chain CDR 3 amino acid sequence ofanti-Ly6E antibody clone xLy6E mu9B12 19 DIQMTQSPSSLSASVGDRVTITCHumanized variable light chain FW 1 amino acid sequence ofanti-Ly6E antibody clone hu9B12 v12 20 WYQQKPGKTVKLLIYHumanized variable light chain FW 2 amino acid sequence ofanti-Ly6E antibody clone hu9B12 v12 21 GVPSRFSGSGSGTDYTLTISSLQPEDFATYYCHumanized variable light chain FW 3 amino acid sequence ofanti-Ly6E antibody clone hu9B12 v12 22 FGQGTKVEIKHumanized variable light chain FW 4 amino acid sequence ofanti-Ly6E antibody clone hu9B12 v12 23 EVQLVESGPALVKPTQTLTLTCTVSHumanized variable heavy chain FW 1 amino acid sequence of anti-Ly6Eantibody clone hu9B12 v12 24 WIRQPPGKALEWLG Humanized variable heavychain FW 2 amino acid sequence of anti-Ly6E antibody clone hu9B12 v12 25RLTISKDTSKNQVVLTMTNMDPVDTATYYCAR Humanized variable heavychain FW 3 amino acid sequence of anti-Ly6E antibody clone hu9B12 v12 26WGQGTLVTVSS Humanized variable heavy chain FW 4 amino acidsequence of anti-Ly6E antibody clone hu9B12 v12 27DIQMTQTTSSLSASLGDRVTISC Chimeric variable light chainFW 1 amino acid sequence of anti-Ly6E antibody clone xLy6E mu9B12 28WYQQKPDGTVKLLIY Chimeric variable light chainFW 2 amino acid sequence of anti-Ly6E antibody clone xLy6E mu9B12 29GVPSRFSGSGSGTDYSLTISNLEPEDIATYYC Chimeric variable light chainFW 3 amino acid sequence of anti-Ly6E antibody clone xLy6E mu9B12 30FGGGTKVEIK Chimeric variable light chain FW 4 amino acid sequence ofanti-Ly6E antibody clone xLy6E mu9B12 31 QVQLKESGPGLVAPSQSLSLTCTVSChimeric variable heavy chain FW 1 amino acid sequence ofanti-Ly6E antibody clone xLy6E mu9B12 32 WVRQPPGKGLEWLGChimeric variable heavy chain FW 2 amino acid sequence ofanti-Ly6E antibody clone xLy6E mu9B12 33RLTISKDNSKSQVFLKMNSLQTDDTARYYCAR Chimeric variable heavy chainFW 3 amino acid sequence of anti-Ly6E antibody clone xLy6E mu9B12 34WGPGTLVTVSA Chimeric variable heavy chain FW 4 amino acid sequence ofanti-Ly6E antibody clone xLy6E mu9B12 35MKIFLPVLLAALLGVERASSLMCFSCLNQKSNL RHESUS Ly6E amino acidYCLKPTICSDQDNYCVTVSTSAGIGNLVTFGHS sequence with signal sequenceLSKTCSPACPLPEGINVGVASMGISCCQSFLCNF (amino acids 1-20, underlined)SAADGGLRASATLLGAGLLLSLLPALLRFGP 36 MSATSNMRVFLPVLLAALLGMEQVHSLMCFSCMOUSE Ly6E amino acid TDQKNNINCLWPVSCQEKDHYCITLSAAAGFGsequence with signal sequence NVNLGYTLNKGCSPICPSENVNLNLGVASVNSY(amino acids 1-26, underlined) CCQSSFCNFSAAGLGLRASIPLLGLGLLLSLLALL QLSP37 MSAASSMRVFLPVLLAALLGVEQVHSLMCFSCTD RAT Ly6E amino acidQKNNINCLWPVSCSSTDNYCITLSAAAGFGNVNL sequence with signal sequenceGYTLNKGCSPTCPRENININLGVASVNSYCCQSSF (amino acids 1-26, underlined)CNFSTAGLGLRASIPLLGLGLLLSLLAVLRLSP 38 LMCFSCLNQKSNLYCLKPTICSDQDNYCVTVSAMature HUMAN Ly6E amino SAGIGNLVTFGHSLSKTCSPACPIPEGVNVGVASacid sequence (without signal MGISCCQSFLCNFSAADGGLRASVTLLGAGLLLsequence) SLLPALLRFGP 39 LMCFSCLNQKSN Mature CYNOMOLOGOUSLYCLKPTICSDQDNYCVTVSTSAGIGNLVTFG Ly6E amino acid sequenceHSLSKTCSPACPLPEGINVGVASMGISCCQSFL (without signal sequence)CNFSAADGGLRASATLLGAGLLLSLLPALLRFGP 40 LMCFSCLNQKSNLMature RHESUS Ly6E amino YCLKPTICSDQDNYCVTVSTSAGIGNLVTFGHSacid sequence (without signal LSKTCSPACPLPEGINVGVASMGISCCQSFLCNFsequence) SAADGGLRASATLLGAGLLLSLLPALLRFGP 41 LMCFSCMature MOUSE Ly6E amino TDQKNNINCLWPVSCQEKDHYCITLSAAAGFGacid sequence (without signal NVNLGYTLNKGCSPICPSENVNLNLGVASVNSYsequence) CCQSSFCNFSAAGLGLRASIPLLGLGLLLSLLALL QLSP 42 LMCFSCTDMature RAT Ly6E amino acid QKNNINCLWPVSCSSTDNYCITLSAAAGFGNVNLsequence (without signal GYTLNKGCSPTCPRENININLGVASVNSYCCQSSF sequence)CNFSTAGLGLRASIPLLGLGLLLSLLAVLRLSP 43 EVQLVESGGGLVQPGGSLRLSCAASGFSLTGYSVNHumanized variable heavy WVRQAPGKGLEWVGMIWGDGSTDYNSALKSRFTchain amino acid sequence of ISRDNSKNTLYLQMNSLRAEDTAVYYCARDYYFNhu9B12 VH3 graft YASWFAYWGQGTLVTVSS 44QVQLKESGPGLVAPSQSLSLTCTVSGFSLTGYSVN Chimeric variable heavy chainWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLT amino acid sequence of xLy6EISKDNSKSQVFLKMNSLQTDDTARYYCARDYYFN mu9B12 in VH3 graftYASWFAYWGPGTLVTVSA 45 DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYHuman kappa I consensus light QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTchain variable amino acid LTISSLQPEDFATYYCQQYYSYPFTFGQGTKVEIK sequence46 EVQLVESGPALVKPTQTLTLTCTFSGFSLSTSGVG Human VH2 consensus heavyVSWIRQPPGKALEWLALIDWNDDKRYSTSLKSRL chain variable amino acidTISKDTSKNQVVLTMTNMDPVDTATYYCARDTA sequence AYFDYWGQGTLVTVSS 47EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMS VH3 consensus heavyWVRQAPGKGLEWVGAISSSGSSTYYADSVKGRFT chain variable amino acidISRDNSKNTLYLQMNSLRAEDTAVYYCARFDYWG sequence QGTLVTVSS

III. EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1 Human Ly6E Gene Expression

GeneLogic Profile: For the analysis of Ly6E mRNA expression in multiplehuman tumor and normal biopsy samples, the Affymetrix data were obtainedfrom Gene Logic Inc. The analysis shown for probe set ID 202145_ at wascarried out using the HGU133 Plus v2 GeneChip on 3,879 normal humantissue samples (green symbols), 1,605 human cancer tissue samples (redsymbols: 1,291 primary and 314 metastatic), and 3,872 human noncancerdisease tissue samples (blue symbols). Microarray data were normalizedusing the Affymetrix MAS (Microarray Analysis Suite) version 5.0software, with sample expression values scaled to a trimmed mean of 500.

This analysis showed that Ly6E was specifically over-expressed inbreast, pancreatic, colon, lung and ovarian cancers with low/nodetection of Ly6E in normal tissues (FIG. 2).

In Situ Hybridization (ISH): In situ hybridization was performed onovarian cancer tissue microarray (TMA) for evaluating prevalence of Ly6Eper methods established in Methods Mol Biol, 2006; 326:255-64.

Forward primer  SEQ ID NO: 48 GTG CCT GAT CTG TGC CCT TGG Reverse primer  SEQ ID NO: 49 CCC GGA AGT UGC AGA AAC CC Probe sequence: SEQ ID NO: 50GTGCCTGATCTGTGCCCTTGGTCCCAGGTCAGGCCCACCCCCTGCACCTCCACCTGCCCCAGCCCCTGCCTCTGCCCAAGTGGGCCAGCTGCCCTCACTTCTGGGGTGGATGATGTGACCTTCCTTGGGGGACTGCGGAAGGGACGAGGGTTCCCTGGAGTCTTACGGTCCAACATCAGACCAAGTCCCATGGACATGCTGACAGGGTCCCCAGGGAGACCGTGTCAGTAGGGATGTGTGCCTGGCTGTGTACGTGGGTGTGCAGTGCACGTGAGAGCACGTGGCGGCTTCTGGGGGCCATGTTTGGGGAGGGAGGTGTGCCAGCAGCCTGGAGAGCCTCAGTCCCTGTAGCCCCCTGCCCTGGCACAGCTGCATGCACTTCAAGGGCAGCCTTTGGGGGTTGGGGTTTCTGCCACTTCCGGG

The results indicated that 47/65 (72%) of ovarian tumors analyzed showedLy6E expression (data not shown).

Immunohistochemistry (IHC): Immunohistochemistry was performed on 4 μmthick formalin-fixed paraffin embedded (FFPE) tissue sections mounted onglass slides. Slides were deparaffinized in xylene and rehydratedthrough graded alcohols to distilled water. Slides were pretreated withTarget Retrieval solution (Dako, Carpinteria, Calif., USA) for 20minutes at 99° C. Slides were then treated with KPL blocking solution(Kierkegaard and Perry Laboratories, Gaithersburg, Md., USA) andavidin/biotin block (Vector Laboratories, Burlingame, Calif., USA)respectively. Non-specific IgG binding was blocked with 10% horse serum(Life Technologies, Carlsbad, Calif., USA) in 3% bovine serum albumin(Roche, Basel, Switzerland) in phosphate buffered saline. Primaryantibody, mouse anti-Ly6E, clone 10G7.7.8 (see Example 3) was diluted 10μg/mL and incubated on slides for 60 minutes at room temperature.

Slides were rinsed, incubated with horse anti-mouse IgG biotinylatedsecondary (Vector Labs) followed by incubation in Vectastain ABC Elitereagent (Vector Labs). Slides were then incubated in Pierce metalenhanced DAB (Thermo Scientific; Fremont, Calif.), counterstained,dehydrated and coverslipped.

From IHC studies, the prevalence of Ly6E was detected at 27-36% inbreast cancer, ˜40% in pancreatic cancer, ˜26% in colon cancer, 17-26%in melanoma, ˜29% in NSCLC (data not shown).

Immunohistochemistry on Normal Tissues: On a panel of normal human andcynomolgus monkey tissues, low and moderate Ly6E expression is detectedin the stomach and salivary glands of both human and cynomolgus monkey,low to moderate Ly6E expression is detected in a subpopulation of cellsin the adrenal cortex in cynomolgus monkey and to a lesser extent inhuman specimens and moderate expression of Ly6E is detected in thetransitional epithelium of the urinary bladder (only cynomolgus monkeywas examined). Table 2 below tabulates immunohistochemical (IHC)expression of Ly6E in a comprehensive human and cynomolgus monkey normaltissue panel. Low (LOW) to moderate (MOD) Ly6E expression is limited tothe underlined tissues (adrenal cortex, cervix, salivary glands, stomachand urinary bladder). ND=not done. NO=no expression.

TABLE 2 Normal Tissue Human Cyno Abdominal Cavity ND NO Adrenal LOW MOD(1/3) Bone Marrow NO NO Brain NO NO Breast NO NO Cervix MOD NO (1/3)Colon NO NO Esophagus NO NO Eye NO NO Heart NO NO Intestine Small NO NOKidney NO NO Larynx NO NO Liver NO NO Lung NO NO Pancreas NO NOParathyroid NO ND Pituitary ND NO Prostate NO NO Salivary Gland MOD MODSkeletal Muscle NO NO Skin NO NO Spleen NO NO Stomach LOW LOW Testis NONO Thymus NO NO Thyroid NO NO Tonsil NO NO Urinary Bladder ND MOD UterusNO NO

Example 2 Quantitative PCR (QRT PCR)

Human major tissue qPCR Array containing 1st strand DNA from a panel of48 normal tissues from Origene, Rockville, Md. (HMRT 102) was assayedfor Ly6E RNA expression. Ly6E expression in a panel of select cancercell lines and tissues (breast and pancreatic) were assayed in parallel.Taqman assays were set up using reagents, instrumentation and softwarefrom Applied Biosystems (ABI, Foster City, Calif.). Primer-probe setswere designed with primers flanking a fluorogenic probe dual labeledwith Reporter dye FAM and quencher dye TAMRA.

Primer-probe set for RPL19:

(SEQ ID NO: 51) Forward primer-5′ AGC GGA TTC TCA TGG AAC A; (SEQ ID NO: 52) Reverse primer-5′ CTG GTC AGC CAG GAG CTT  and (SEQ ID NO: 53) probe-5′ TCC ACA AGC TGA AGG CAG ACA AGG.Primer-probe set for Ly6E: (SEQ ID NO: 54) Forward primer-5′AGA AGG CGT CAA TGT TGG T;  (SEQ ID NO: 55) Reverse primer-5′CAC TGA AAT TGC ACA GAA AGC and  (SEQ ID NO: 56) probe-5′TTC CAT GGG CAT CAG CTG CTG.

The results indicate that the Ly6E transcript expression in normaltissues is low compared to expression of Ly6E in breast and pancreaticcancers (FIG. 3).

Example 3 Antibody Generation and Humanization

For production of anti-Ly6E monoclonal antibodies, clones 4D8, 10G7 and9B12, 5 female BALB/c mice were immunized with either bacterial(Escherichia Coli) generated His tagged Ly6E or mammalian (CHO-K1S)generated C-Term myc 6× His tagged Ly6E protein as follows:

Generation of Human Ly6E cDNA: Human Ly6E from Origene, Rockville, Md.and Cynomolgus monkey Ly6E cDNA from Open Biosystems, Lafayette, Colo.were cloned into a retroviral N-terminal gD-tagged vector. Theseconstructs were used to generate pools of PC3 cells stably expressinghuman and Cynomolgus monkey Ly6E, respectively.

In addition, His tag Human Ly6E was cloned into a CMV promoter drivenmammalian expression system and into a bacterial expression system togenerate secreted protein from CHO-K1 suspension cells and fromEscherichia Coli.

Immunization of Mice: Mice were immunized with 6 bi-weekly foot padinjections of 2 μg protein re-suspended in monophosphoryl lipidA/trehalose dicorynomycolate adjuvant (Ribi Immunochemicals). Three daysafter the final boost, popliteal lymph node cells were fused with cellsderived from the murine myeloma cell line P3X63AgU.1 (CRL1597; AmericanType Culture Collection) using 50% polyethylene glycol. Hybridomas wereselected using hypoxanthineaminopterin-thymidine (HAT) medium in 96-wellplates. Ten to 14 days later, culture supernatants were collected andscreened by direct ELISA against the immunogen and by flow cytometricanalysis for binding to Ly6E on HT1080 transfected cell lines and thensub-cloned by limiting dilution.

Generation of hu9B16 CDR grafts—Several CDR grafts of murine 9B12(mu9B12) were generated by Kunkel mutagenesis, using a separateoligonucleotide for each hypervariable region. The constructs were madein the context of transient IgG expression vectors. Correct clones wereassessed by DNA sequencing. IgG was expressed and purified as described(See Liang, W.-C. et al. Function blocking antibodies to neuropilin-1generated from a designed human synthetic antibody phage library.Journal of Molecular Biology 366, 815-829 (2007)).

Cell-based Ly6E competitive binding assay—Cultured human Ly6Etransfected PC3 cells were harvested with 5 mM EDTA containing PBS.Cells were washed with PBS and added onto a 384-well high binding plate(Meso Scale Discovery Technology (MSD); Gaithersburg, Md.). The platewas kept at room temperature for 1 hr to allow cells to adhere to theplate as described (See Lu, Y., Wong, W. L. & Meng, Y. G. A highthroughput electrochemiluminescent cell-binding assay for therapeuticanti-CD20 antibody selection. Journal of Immunological Methods 314,74-79 (2006)). The plate with cells was blocked with fetal bovine serumcontaining PBS for 1 hr and then cooled on ice. Serially dilutedantibody variant samples were mixed with equal volume of fixedconcentration of mouse 9B12 Ab. The mixtures were added onto the platesand incubated at 4° C. with gentle shaking for 1 hr.

The plate was then washed with cold PBS and an anti-mouse IgG Fcspecific Ab (Jackson ImmunoReserach; West Grove, Pa.) labeled withsulfo-ruthenium tag was added to the plate as the detection reagent. Theplate was incubated at 4° C. with gentle shaking for 1 hr. Afterincubation the plate was washed again, and MSD read buffer (MSD;Gaithersburg, Md.) was added onto the wells. The plate was read with aMSD SectorÒ imager 6000. The data was graphed and analyzed usingKaleidaGraph software (Synergy Software; Reading, Pa.) to determine IC₅₀values.

SPR Affinity Determination—Affinity determinations were performed bysurface plasmon resonance using single cycle kinetics on aBIAcore™-T100. Hu Ly6E was immobilized via EDC/NHS chemistry accordingto supplier's instructions (˜20 response units (RU)) on a CM5 chip. Forkinetic measurement, three-fold serial dilution of anti-Ly6E IgG (5 to405 nM) in PBST were injected with 200 s for association & 300 s fordissociation at a flow rate of 30 ul/min at 25° C. Binding response wascorrected by subtracting both the RU from a blank flow cell and frombuffer run on the same flow cell. A 1:1 Languir model of simultaneousfitting of k_(on) and k_(off) was used to measure the apparent KD.

Humanization of 9B12 (anti-Ly6E)

In order to humanize murine 9B12, the hypervariable regions (HVRs) weregrafted into either a kappa I-VH₂ or kappa I-VH₃ consensus framework togenerate several CDR grafts containing different combinations ofpotential vernier positions. Variable domain sequences of 9B12 variantsaligned with human consensus (FIG. 4) kappa I and (FIG. 5) VH₂ or (FIG.6) VH₃ variable domain frameworks Amino acid positions that differ fromthe human consensus frameworks are highlighted in grey; regions thatwere transferred to generate the CDR graft are boxed. Positions arenumbered according to Kabat (See Kabat, E. A., Wu, T. T., Perry, H. M.,Gottesman, K. S. & Foeller, C. Sequences of proteins of immunologicalinterest, Edn. 5th. (Public Health Service, National Institutes ofHealth, Bethesda, Md.; 1991). The HVR regions used in the VL domainwere: positions 24-34 (L1), 50-56 (L2) and 89-97 (L3) (FIG. 4). In theVH domain, positions 26-35 (H1), 50-65 (H2) and 95-102 (H3) were grafted(FIGS. 5 and 6).

Several positions that potentially influence HVR conformation (vernierpositions) in the new human framework were reverted to the mousesequence in an effort to explore their role on Ly6E binding affinity. Inthe kappa I domain, combinations of positions 43, 44, and 71 wereincluded. In the VH₂ domain, combinations of positions 24, 37, 49, 73and 76 were explored while in the VH₃ domain, combinations of positions24, 48, 67, 71, 76 and 78 were tested. All together 25 variants wereconstructed and expressed as IgG (Table 3). Purtified IgG were thenscreened in a cell-based Ly6E competitive binding assay. Most HVRconstructs bound to Ly6E regardless of whether the consensus VH₂ or VH₃variable heavy domain was used. The best clone with the fewestadditional changes, hu9B12.v12, was obtained using the VH₂ domain andcontained 3 vernier positions in kappa I (43, 44 and 71) and 2 in VH₂(24 and 49). Table 3 shows a matrix of humanized HVR framework-repairvariants that were constructed and assessed using the cell-based Ly6Ecompetitive binding assay.

TABLE 3 Light Chain k1 graft k1 graft k1 graft + k1 graft 43 + 44 43 +44 + 71 71 Heavy Chain VH2 graft VH2 graft + 49 v1 (no binding) v6 (nobinding) v11 (no binding) VH2 graft + 24 + 49 v2 (28, 37 nM) v7 (24, 36nM) v12 (15, 16 nM) VH2 graft + 24 + 37 + 49 v16 (27 nM) v21 (82 nM) VH2graft + 24 + 49 + 73 v17 (29 nM) v22 (59 nM) VH2 graft + 24 + 49 + 73 +76 v18 (22 nM) v23 (54 nM) VH2 graft + 24 + 37 + 49 + 73 + 76 v19 (28nM) v24 (53 nM) VH3 graft v3 (no binding) v8 (no binding) v13 (nobinding) VH3 graft + 24 v4 (98, 70 nM) v9 (34, 44 nM) v14 (39, 56 nM)VH3 graft + 71 + 78 v5 (70 nM) v10 (61 nM) v15 (42, 52 nM) VH3 graft +24 + 48 + 67 + 71 + 76 + 78 v20 (26 nM) v25 (56 nM) chimeric 9B12 (3, 6,5 nM)

This clone had about 3-5-fold reduce affinity for human Ly6E in thecell-based Ly6E competitive binding assay but similar affinity by SPRand scatchard analysis (Table 4), where ch9B12 denotes the chimericvariant and 9B12.v12 denotes the humanized variant.

TABLE 4 cell-based Ly6E competitive Scatchard Analysis Biacore bindingassay cyno hu Ly6E cyno hu Ly6E Ly6E KD hu Ly6E Ly6E KD (nM) KD (nM)(nM) KD (nM) KD (nM) Ch 9B12 4 4 6 3-6 5-7 9B12.v12 4 14 7 15-19 16-40

Example 4 Binding of Anti-Ly6E Antibody to Human and Cynomologous Ly6E

A scatchard analysis was performed to determine affinity and bindingsites per cell for the antibodies. Hu.9B12v12 antibody was iodinatedseveral times using the Iodogen method. The radiolabeled Hu.9B12v12antibody was purified from free ¹²⁵I-Na by gel filtration using a NAP-5column and had a range of specific activity of 11.14-16.01 μCi/m.Competition reaction mixtures of 50 μL containing a fixed concentrationof iodinated antibody and decreasing concentrations of unlabeledantibody were placed into 96-well plates. The PC3 cells stablytransduced with retrovirus to express either recombinant human orCynomolgus monkey gD tagged Ly6E were detached from flasks using SigmaCell Dissociation Solution and were washed with binding buffer (DMEMwith 2% FBS, 50 mM HEPES, pH 7.2, and 0.1% sodium azide). The washedcells were added at an approximate density of 200,000 cells in 0.2 mL ofbinding buffer to the 96-well plates containing the 50-μL competitionreaction mixtures. The final concentration of the iodinated antibody ineach competition reaction with cells was 200 pM and the finalconcentration of the unlabeled antibody in the competition reaction withcells varied, starting at 500 nM and then decreasing by 1:2-folddilution for ten concentrations, and included a zero-added, buffer-onlysample. Competition reactions with cells for each concentration ofunlabeled antibody were assayed in triplicate. Competition reactionswith cells were incubated for 2 hours at room temperature. After the2-hour incubation, the competition reactions were transferred to aMillipore Multiscreen filter plate and washed four times with bindingbuffer to separate the free from bound iodinated antibody. The filterswere counted on a Wallac Wizard 1470 gamma counter (PerkinElmer Life andAnalytical Sciences; Wellesley, Mass.). The binding data were evaluatedusing New Ligand software (Genentech), which uses the fitting algorithmof Munson and Rodbard (1980) to determine the binding affinity of theantibody. As shown in FIG. 6, Panel A and B and in Table 5, the bindingaffinity of Hu9B12v12 on human and cynomolgus monkey was estimated at4.0 nM and 7.9 nM respectively.

TABLE 5 Antibody Species Affinity Sites/Cell Ch.9B12 Human   2 nM 7000Cyno 2.9 nM 16,000 Hu.9B12.v12 Human   4 nM 7000 Cyno 7.9 nM 16,000 gDHuman 1.1 nM 13,000 Cyno   2 nM 53,000

Example 5 Generation of Antibody Drug Conjugates

For larger scale antibody production, antibodies were produced in CHOcells. Vectors coding for VL and VH were transfected into CHO cells andIgG was purified from cell culture media by protein A affinitychromatography.

Generation of vcMMAE ADC: Anti-Ly6E antibody-drug conjugates (ADCs) wereproduced by conjugating hu9B12.v12 or control anti gD ADCs wereconjugated to the drug-linker moiety MC-vc-PAB-MMAE, which is depictedherein. For convenience, the drug-linker moiety MC-vc-PAB-MMAE issometimes referred to in these Examples and in the Figures as “vcMMAE”or “VCE.” Prior to conjugation, the antibodies were partially reducedwith TCEP using standard methods in accordance with the methodologydescribed in WO 2004/010957 A2. The partially reduced antibodies wereconjugated to the drug-linker moiety using standard methods inaccordance with the methodology described, e.g., in Doronina et al.(2003) Nat. Biotechnol. 21:778-784 and US 2005/0238649 A1. Briefly, thepartially reduced antibodies were combined with the drug-linker moietyto allow conjugation of the drug-linker moiety to reduced cysteineresidues of the antibody. The conjugation reactions were quenched, andthe ADCs were purified. The drug load (average number of drug moietiesper antibody) for each ADC was determined and was between 3.3 and 4.0for the anti-Ly6E antibodies and anti-gD control antibodies.

Example 6 Binding of Humanized Anti-Ly6E ADC to Human and Cyno Ly6E

In vitro killing assay: To assess the effects of Hu9B12v12-ADC on cellviability, cells were plated at 1,500 per well in 50 μL of normal growthmedium in 96-well clear-bottom black plates. Twenty-four hours later, anadditional 50 μL of culture medium with serial dilutions ofHu9B12v12-ADC concentrations was added to triplicate wells. Five dayslater, cell survival was determined using CellTiter-Glo Luminescent CellViability Reagent (G7572; Promega Corporation) and with an EnVision 2101Mutilabel Reader (Perkin-Elmer). For the two cell lines tested, in vitrokilling efficacy appeared proportional to the expression of Ly6E on thecell surface (FIG. 6, Panels A and B).

Flow Cytometry: For fluorescence-activated cell sorting (FACS), cellswere harvested in PBS with 2.5 mmol/L EDTA and washed in PBS buffercontaining 1% FBS. All subsequent steps were carried out at 4° C. Cellswere incubated for 1 hour each with 3 to 5 μg/mL primary antibodies,followed by the appropriate secondary antibodies. Cells were thenanalyzed with a FACS Calibur flow cytometer (BD Biosciences) and GeoMeanvalues were obtained. Primary antibodies, Hu.9B12v12 for Ly6E cellsurface detection, in-house generated anti-gD mAb for N-Term gD tagdetection were used. Alexa 488-conjugated anti-mouse or anti-human IgGfluorescent detection reagent (A11017, A11013; Invitrogen) were used.

Example 7 In Vivo Efficacy of Anti-Ly6E ADC in Xenograft Mouse Model

Breast cancer cell line, HCC1569 (CRL-2330), pancreatic cancer cell lineSU.86.86 (CRL-1837), Chinese Hamster ovary cell line, CHO-K1 (CC1-61)and prostatic cancer cell line PC3 (CRL-1435) were obtained fromAmerican Type Culture Collection (ATCC, Manassas, Va.). CHO-K1S is asuspension cell line derivative of CHO-K1. The HCC1569 X2 cell line is aderivative of the parental HCC1569 cell line (ATCC, CRL-2330) optimizedfor growth in vivo. Parental HCC1569 cells were injected subcutaneouslyin the right flank of female Taconic NCr nude mice, one tumor washarvested, minced and grown in vitro resulting in the HCC1569 X1 cellline. The HCC1569 X1 line was injected again subcutaneously in the rightflank of female Taconic NCr nude mice in an effort to improve the growthof the cell line. A tumor from this study was collected and againadapted for in vitro growth to generate the HCC1569 X2 cell line. Thiscell line and tumors derived from this line express Ly6E.

Xenograft models: Efficacy of anti-Ly6E antibody drug conjugates (ADCs)was evaluated in xenograft models derived from cell lines describedabove or in primary patient derived tumor models, the latter experimentswere conducted at Oncotest, Freiburg, Germany and in XenTech, Genopole,France.

All studies conducted at Genentech, South San Francisco, Calif. were inaccordance with the Guide for the Care and Use of Laboratory Animals(Ref: Institute of Laboratory Animal Resources (NIH publication no.85-23), Washington, D.C.: National Academies Press; 1996). Allexperiments conducted at Oncotest were approved by the localauthorities, and are conducted according to the guidelines of the GermanAnimal Welfare Act (Tierschutzgesetz). The authorization to use animalsin the CERFE facilities of XenTech was obtained by The Direction desServices Vétérinaires, Ministère de l'Agriculture et de la Pêche, France(agreement No. A 91-228-107). The animal care and housing are inaccordance with European Convention STE 123. All experiments at XenTechwill be performed in accordance with French legislation concerning theprotection of laboratory animals and in accordance with a currentlyvalid license for experiments on vertebrate animals, issued by theFrench Ministry for Agriculture and Fisheries to Dr. Truong-An TRAN (No.A 91-541 dated 21 Dec. 2010; validity: 5 years). 6- to 9-week old femaleimmunodeficient mice were inoculated subcutaneously in the dorsal rightflank and average tumor volumes with SDs were determined from 9-10 miceper group.

For efficacy studies with xenografts derived from cell lines, NCR nudemice from Taconic were inoculated with 5 million cells in HBSS withMatrigel or C.B-17 SCID (inbred) mice From Charles River were inoculatedwith 2 million cells in HBSS with Matrigel. 0.36 mg estrogen implantswere used for the HCC1569 X2 xenograft model. For efficacy studies withtumor explants at XenTech and Oncotest, athymic nude or NMRI nu/nu micefrom Harlan or Charles River were implanted with primary breast orpancreatic cancer patient derived materials from models HBCx-8, HBCx-9,MAXF-1162 and PAXF-1657. When tumor volumes reached approximately 80-200mm3 (day 0), animals were randomized into groups of 9-10 each andadministered a single intravenous (IV) injection of either vehiclecontrol or the ADC at the appropriate dose. Tumor volumes were measuredtwice per week until study end.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1.-56. (canceled)
 57. A method of treating an individual having anLy6E-positive cancer, the method comprising administering to theindividual an effective amount of an immunoconjugate comprising anisolated monoclonal antibody that binds to Ly6E and a cytotoxic agent,wherein the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:10; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:11; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO:12; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:7; (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:9.
 58. The method ofclaim 57, wherein the Ly6E-positive cancer is selected from a breastcancer, pancreatic cancer, colon cancer, colorectal cancer, melanoma,ovarian cancer, non-small cell lung cancer, or gastric cancer.
 59. Themethod of claim 57, further comprising administering an additionaltherapeutic agent to the individual.
 60. The method of claim 57, whereinthe additional therapeutic agent is a platinum complex.
 61. The methodof claim 57, wherein the immunoconjugate has the formula Ab-(L-D)p,wherein: (a) Ab is the antibody; (b) L is a linker; (c) D is a cytotoxicagent selected from a maytansinoid, an auristatin, a calicheamicin, apyrrolobenzodiazepine, and a nemorubicin derivative; and (d) p rangesfrom 1-8.
 62. The method of claim 57, wherein the cytotoxic agent is anauristatin.
 63. The method of claim 61, wherein D has formula D_(E)

and wherein R² and R⁶ are each methyl, R³ and R⁴ are each isopropyl, R⁵is H, R⁷ is sec-butyl, each R⁸ is independently selected from CH₃,O—CH₃, OH, and H; R⁹ is H; and R¹⁸ is —C(R⁸)₂—C(R⁸)₂-aryl.
 64. Themethod of claim 57, wherein the cytotoxic agent is MMAE.
 65. The methodof claim 57, wherein the cytotoxic agent is a pyrrolobenzodiazepine. 66.The method of claim 61, wherein D is a pyrrolobenzodiazepine of FormulaA:

wherein the dotted lines indicate the optional presence of a double bondbetween C1 and C2 or C2 and C3; R² is independently selected from H, OH,═O, ═CH₂, CN, R, OR, ═CH—R^(D), ═C(R^(D))₂, O—SO₂—R, CO₂R and COR, andoptionally further selected from halo or dihalo, wherein R^(D) isindependently selected from R, CO₂R, COR, CHO, CO₂H, and halo; R⁶ and R⁹are independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo; R⁷ is independently selected from H, R, OH, OR, SH,SR, NH₂, NHR, NRR′, NO₂, Me₃Sn and halo; Q is independently selectedfrom O, S and NH; R¹¹ is either H, or R or, where Q is O, SO₃M, where Mis a metal cation; R and R′ are each independently selected fromoptionally substituted C₁₋₈ alkyl, C₃₋₈ heterocyclyl and C₅₋₂₀ arylgroups, and optionally in relation to the group NRR′, R and R′ togetherwith the nitrogen atom to which they are attached form an optionallysubstituted 4-, 5-, 6- or 7-membered heterocyclic ring; R¹², R¹⁶, R¹⁹and R¹⁷ are as defined for R², R⁶, R⁹ and R⁷ respectively; R″ is a C₃₋₁₂alkylene group, which chain may be interrupted by one or moreheteroatoms and/or aromatic rings that are optionally substituted; and Xand X′ are independently selected from O, S and N(H).
 67. The method ofclaim 61, wherein D has the structure:

wherein n is 0 or
 1. 68. The method of claim 57, wherein the cytotoxicagent is a nemorubicin derivative.
 69. The method of claim 61, wherein Dhas a structure selected from:


70. The method of claim 61, wherein the linker is cleavable by aprotease.
 71. The method of claim 61, wherein the linker comprises avaline-citrulline, alanine-phenylalanine, phenylalanine-lysine,phenylalanine-homolysine, or N-methyl-valine-citrulline dipeptide. 72.The method of claim 61, wherein the linker is acid-labile.
 73. Themethod of claim 72, wherein the linker comprises hydrazone.
 74. Themethod of claim 61 having the formula:

wherein S is a sulfur atom.
 75. The method of claim 61 having theformula:


76. The method of claim 61 having a formula selected from:

wherein R₁ and R₂ are independently selected from H and C₁-C₆ alkyl; and


77. The method of claim 61, wherein p ranges from 2-5.
 78. The method ofclaim 74, wherein the antibody comprises a VH sequence of SEQ ID NO:5and a VL sequence of SEQ ID NO:3.
 79. A method of treating an individualhaving an Ly6E-positive cancer, the method comprising administering tothe individual an effective amount of an immunoconjugate having theformula:

wherein a. Ab is a monoclonal antibody that binds to Ly6E, wherein saidantibody comprises (i) HVR-H1 comprising the amino acid sequence of SEQID NO:10; (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:11; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:12;(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:7; (v)HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; (vi) HVR-L3comprising the amino acid sequence of SEQ ID NO:9; b. S is a sulfuratom; and c. p ranges from 2-5.